+

WO2001066738A2 - Acides nucleiques codant des polypeptides crsp1 humains et utilisations de ces derniers - Google Patents

Acides nucleiques codant des polypeptides crsp1 humains et utilisations de ces derniers Download PDF

Info

Publication number
WO2001066738A2
WO2001066738A2 PCT/US2001/003685 US0103685W WO0166738A2 WO 2001066738 A2 WO2001066738 A2 WO 2001066738A2 US 0103685 W US0103685 W US 0103685W WO 0166738 A2 WO0166738 A2 WO 0166738A2
Authority
WO
WIPO (PCT)
Prior art keywords
hcrspl
polypeptide
nucleic acid
seq
fragment
Prior art date
Application number
PCT/US2001/003685
Other languages
English (en)
Other versions
WO2001066738A3 (fr
Inventor
Eric Wen Su
Original Assignee
Eli Lilly And Company
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eli Lilly And Company filed Critical Eli Lilly And Company
Priority to AU2001239742A priority Critical patent/AU2001239742A1/en
Publication of WO2001066738A2 publication Critical patent/WO2001066738A2/fr
Publication of WO2001066738A3 publication Critical patent/WO2001066738A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • the present invention relates to compounds and compositions comprising novel human cysteine-rich secreted protein (hCRSP) polypeptides, nucleic acids, host cells, transgenics, chimerics, antibodies, compositions, and methods of making and using thereof.
  • hCRSP human cysteine-rich secreted protein
  • Matricellular proteins comprise a non-homologous group of extra-cellular regulatory macromolecules that mediate cell matrix interactions but may not contribute significantly to extra-cellular matrix structure (Bornstein, P., J. Cell Biol., 130:503-506(1995)).
  • the matricellular class of secreted glyco-proteins includes SPARC, thrombospondins 1 and 2, tenascins C and X, and osteopontin. Although structurally unrelated, these molecules appear to perform related functions, e.g., they exhibit counter- adhesive effects that lead to cell rounding and changes in cell shape that result in the disruption of cell-matrix interactions .
  • SPARC is the prototype of the matricellular proteins. While SPARC is expressed at high levels in bone tissue, it is also distributed widely in other tissues and cell types (Maillard, C, et al . , Bone, 13:257-267 (1992)).
  • SPARC is associated generally with remodeling tissues, e.g., tissues undergoing morphogenesis, mineralization, angiogenesis, tumorigenesis, and pathological responses to injury.
  • remodeling tissues e.g., tissues undergoing morphogenesis, mineralization, angiogenesis, tumorigenesis, and pathological responses to injury.
  • Experiments in vi tro have also identified SPARC in tumors (Schulz, A., et al., Am. J. Pathol . , 132:233-238 (1988); Porter, P.L., et al . , J. Histochem. Cytoche . , 43:791-800
  • the present invention provides isolated nucleic acids and hCRSPl polypeptides encoded thereby, including specified fragments and variants thereof, as well as hCRSPl compositions, probes, primers, vectors, host cells, antibodies, transgenics, chimerics and methods of making and using thereof, as described and enabled herein.
  • hCRSPl gene Having the cloned hCRSPl gene enables the production of recombinant hCRSPl proteins, the isolation of homologous genes from other organisms, and/or related genes from the same organism, chromosome mapping studies, and the implementation of large scale screens to identify compounds that bind said protein and modulate the activity thereof.
  • the proteins disclosed herein are also useful, among other things, as feed additives.
  • the present invention provides, in one aspect, isolated nucleic acid molecules comprising a polynucleotide encoding hCRSPl polypeptides as shown in SEQ ID NO: 4, or 5 , as well as fragments or specified variants thereof or poly- nucleotides complementary to such hCRSPl polypeptide encoding polynucleotides .
  • the present invention further provides recombinant vectors, comprising 1-40 of said isolated hCRSPl nucleic acid molecules of the present invention, host cells containing such nucleic acids and/or recombinant vectors, as well as methods of making and/or using such nucleic acid, vectors and/or host cells.
  • the present invention also provides methods of making or using such nucleic acids, vectors and/or host cells, such as, but not limited to, using them for the production of hCRSPl nucleic acids and/or polypeptides by known recombinant, synthetic and/or purification techniques, based on the teaching and guidance presented herein in combination with what is known in the art.
  • the present invention also provides an isolated hCRSPl polypeptide comprising at least one fragment, domain, or specified variant with at least 90% identity to at least 20 contiguous amino acids of SEQ ID NO: 4 or 5.
  • the present invention also provides an isolated hCRSPl polypeptide comprising at least one fragment, domain, or specified variant with at least 90% identity to at least 30 contiguous amino acids of SEQ ID NO: 4 or 5.
  • the present invention also provides an isolated hCRSPl polypeptide comprising at least one fragment, domain, or specified variant with at least 90% identity to at least 40 contiguous amino acids of SEQ ID NO: 4 or 5.
  • the present invention also provides an isolated hCRSPl polypeptide comprising at least one fragment, domain, or specified variant with at least 90% identity to at least 50 contiguous amino acids of SEQ ID NO: 4 or 5.
  • the present invention also provides an isolated hCRSPl polypeptide comprising at least one fragment, domain, or specified variant with at least 90% identity to at least 60 contiguous amino acids of SEQ ID NO: 4 or 5.
  • the present invention also provides an isolated hCRSPl polypeptide comprising at least one fragment, domain, or specified variant with at least 90% identity to at least 70 contiguous amino acids of SEQ ID NO: 4 or 5.
  • the present invention also provides an isolated hCRSPl polypeptide as described herein wherein said polypeptide further comprises at least one substitution, insertion, or deletion of at least one portion or residue of SEQ ID NO: 4 or 5.
  • the present invention also provides an isolated polynucleotide comprising a polynucleotide at least 90% identical to at least 100 contiguous nucleotides shown in SEQ ID N0S:1, 2, 3, or a complement thereof.
  • the present invention also provides an isolated polynucleotide comprising a polynucleotide at least 90% identical to at least 90 contiguous nucleotides shown in SEQ ID N0S.-1, 2, 3, or a complement thereof.
  • the present invention also provides an isolated polynucleotide comprising a polynucleotide at least 90% identical to at least 80 contiguous nucleotides shown in SEQ
  • the present invention also provides an isolated polynucleotide comprising a polynucleotide at least 90% identical to at least 70 contiguous nucleotides shown in SEQ ID NOS:l, 2, 3, or a complement thereof.
  • the present invention also provides an isolated polynucleotide comprising a polynucleotide at least 90% identical to at least 70 contiguous nucleotides shown in SEQ
  • the present invention also provides an isolated polynucleotide comprising a polynucleotide at least 90% identical to at least 60 contiguous nucleotides shown in SEQ
  • the present invention also provides an isolated polynucleotide comprising a polynucleotide at least 90% identical to at least 50 contiguous nucleotides shown in SEQ
  • the present invention also provides a recombinant vector comprising an isolated hCRSPl nucleic acid as described herein.
  • the present invention also provides a host cell comprising an isolated hCRSPl nucleic acid as described herein.
  • the present invention also provides a method for constructing a recombinant host cell that expresses an hCRSPl polypeptide, comprising introducing into the host cell an hCRSPl nucleic acid in replicatable form as described herein to provide the recombinant host cell.
  • the present invention also provides a recombinant host cell provided by a method as described herein.
  • the present invention also provides a method for expressing at least one hCRSPl polypeptide in a recombinant host cell, comprising culturing a recombinant host cell as described herein under conditions wherein at least one hCRSPl polypeptide is expressed in detectable or recoverable amounts .
  • the present invention also provides an isolated hCRSPl polypeptide produced by a recombinant, synthetic, and/or any suitable purification method as described herein and/or as known in the art .
  • the present invention also provides an hCRSPl antibody or fragment, comprising a polyclonal and/or monoclonal antibody or fragment that specifically binds at least one epitope specific to at least one hCRSPl polypeptide as described herein.
  • the present invention also provides a method for producing an hCRSPl antibody or antibody fragment, comprising generating the antibody or fragment that binds at least one epitope that is specific to an isolated hCRSPl polypeptide as described herein, the generating done by known recombinant, synthetic and/or hybridoma methods.
  • the present invention also provides an hCRSPl antibody or fragment produced by a method as described herein or as known in the art.
  • the invention also encompasses nucleotide sequences that can be used to inhibit hCRSPl gene expression (e.g., antisense and ribozyme molecules, and gene or regulatory sequence replacement constructs) or to enhance hCRSPl gene expression (e.g., expression constructs that place the hCRSPl gene under the control of a strong promoter system) , and transgenic animals that express hCRSPl transgene or "knock-outs" that do not express hCRSPl.
  • hCRSPl gene expression e.g., antisense and ribozyme molecules, and gene or regulatory sequence replacement constructs
  • enhance hCRSPl gene expression e.g., expression constructs that place the hCRSPl gene under the control of a strong promoter system
  • transgenic animals that express hCRSPl transgene or "knock-outs" that do not express hCRSPl.
  • the present invention also provides an isolated hCRSPl polypeptide as described herein, wherein the polypeptide has at least one activity, such as, but not limited to, promoting or inhibiting angiogenesis, neovascularization, tumorigenesis, cataractogenesis, wound healing, growth- factor mediated chemotaxis, neurite outgrowth and/or neurite adhesion.
  • An hCRSPl polypeptide can thus be screened for a corresponding activity according to methods known in the art (Sage, E.H., and Vernon, R.B., J. Hypertension 12:S145-152 (1995)); Sage, E.H., Adv. Oncol. 12:17-29 (1996); Ledda F., et al., Nature Med.
  • the present invention also provides methods for identifying compounds that bind an hCRSPl polypeptide, comprising a) admixing at least one isolated hCRSPl polypeptide as described herein with a test compound or composition; and b) detecting at least one binding interaction between the polypeptide and the compound or composition, optionally further comprising detecting a change in biological activity attributable herein to hCRSPl, such as a reduction or increase in cellular proliferation.
  • the invention provides a method of screening for compounds that mimic the activity of hCRSPl polypeptides (agonists) or diminish the effect of hCRSPl activity (antagonists) .
  • the present invention also provides a composition comprising an isolated hCRSPl nucleic acid, polypeptide, and/or a antibody of the present invention as described herein and a carrier or diluent.
  • the carrier or diluent can optionally be pharmaceutically acceptable, according to known methods .
  • the present invention provides compounds and pharmaceutical compositions comprising hCRSPl nucleic acids, polypeptides, any fragments or variants thereof, and/or anti-hCRSPl antibodies, for use in methods for treating or preventing tumorigenicity and/or other disorders associated with aberrant hCRSPl activity in mammals in need thereof.
  • the present invention also provides a method of inducing or inhibiting angiogenesis, neovascularization, tumorigenesis, cataractogenesis , wound healing, growth- factor mediated chemotaxis, neurite outgrowth, and/or neurite adhesion in vi tro or in vivo that comprises administering to at least one cell and/or a mammal an effective amount of a composition comprising an isolated hCRSPl nucleic acid, polypeptide, agonist, antagonist, and/or anti-hCRSPl antibody as described herein and a carrier or diluent.
  • the carrier or diluent can optionally be pharmaceutically acceptable, according to known methods.
  • the present invention provides isolated, recombinant and/or synthetic nucleic acid molecules comprising at least one polynucleotide encoding at least one hCRSPl polypeptide comprising specific full length sequences, fragments and specified variants thereof, such polypeptides, and methods of making and using said nucleic acids and polypeptides thereof.
  • An hCRSPl polypeptide of the invention comprises at least one fragment, domain, and/or specified variant of any portion or fragment of any hCRSPl protein as described herein.
  • the present invention also provides at least one utility by providing isolated nucleic acids comprising polynucleotides of sufficient length and complementarity to an hCRSPl nucleic acid for use as probes or amplification primers in the detection, quantitation, or isolation of gene sequences or transcripts.
  • isolated nucleic acids of the present invention can be used as probes for detecting deficiencies in the level of mRNA, in screens for detection of mutations in at least one hCRSPl gene (e.g., substitutions, deletions, or additions), or for monitoring upregulation of expression of said gene, or changes in biological activity as described herein in screening assays of compounds, and/or for detection of any number of allelic variants (polymorphisms or isoforms) of the gene.
  • hCRSPl gene e.g., substitutions, deletions, or additions
  • allelic variants polymorphisms or isoforms
  • the isolated nucleic acids of the present invention can also be used for recombinant expression of hCRSPl polypeptides for use as immunogens in the preparation and/or screening of antibodies.
  • the isolated nucleic acids of the present invention can also be employed for use in sense or antisense suppression of one or more hCRSPl genes or nucleic acids, in a host cell, or tissue in vivo or in vi tro. Attachment of chemical agents which bind, intercalate, cleave and/or crosslink to the isolated nucleic acids of the present invention can also be used to modulate transcription or translation of at least one nucleic acid disclosed herein.
  • an "immunological” activity refers only to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring hCRSPl.
  • a preferred biological hCRSPl activity includes, but is not limited to, induction and/or inhibition of angiogenesis, neovascularization, tumorigenesis, cataractogenesis, rate of wound healing, growth-factor mediated chemotaxis, neurite outgrowth, and/or neurite adhesion.
  • amino acid is used herein in its broadest sense, and includes naturally occurring amino acids as well -lias non-naturally occurring amino acids, including amino acid variants and derivatives. The latter includes molecules containing an amino acid moiety.
  • amino acid includes, for example, naturally occurring proteogenic L-amino acids; D-amino acids; chemically modified amino acids such as amino acid variants and derivatives; naturally occurring non- proteogenic amino acids such as norleucine, ⁇ -alanine, ornithine, etc.; and chemically synthesized compounds having properties known in the art to be characteristic of amino acids .
  • D-CRSPl polypeptides The incorporation of non-natural amino acids, including synthetic non-native amino acids, substituted amino acids, or one or more D-amino acids into the hCRSPl polypeptides and/or fragments thereof and/or variants thereof of the present invention ( "D-CRSPl polypeptides") is advantageous in a number of different ways. D-amino acid-containing polypeptides exhibit increased stability in vi tro ox in vivo compared to L-amino acid-containing counterparts. Thus, the construction of hCRSPl polypeptides incorporating D-amino acids can be particularly useful when greater stability is desired or required in vivo.
  • D-peptides are resistant to endogenous peptidases and proteases, thereby providing improved bioavailability of the molecule, and prolonged lifetimes in vivo when such properties are desirable.
  • the use of L-amino acids therein will permit endogenous peptidases, proteases, etc., to digest the molecule, thereby limiting the cell's exposure to the molecule.
  • D- peptides cannot be processed efficiently for major histocompatibility complex class II-restricted presentation to T hehCRSPler cells, and are therefore less likely to induce humoral immune responses in the whole organism.
  • antagonist is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native hCRSPl polypeptide disclosed herein.
  • agonist is used similarly in the broadest sense and includes any molecule that mimics a biological activity of a native hCRSPl polypeptide disclosed herein.
  • CRSPl agonists and antagonists of the present invention may include, for example, small molecules, hCRSPl polypeptides as defined herein and antibodies directed against hCRSPl polypeptides.
  • Agonists and antagonists of the invention may also include nucleotide sequences, such as antisense and ribozyme molecules, and gene or regulatory sequence replacement constructs, that can be used to inhibit or enhance expression of the hCRSPl gene.
  • Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native hCRSPl polypeptides, peptides, small organic molecules, etc.
  • Methods for identifying agonists or antagonists of an hCRSPl polypeptide may comprise contacting an hCRSPl polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the hCRSPl polypeptide.
  • CRSPl refers to a polynucleotide, or amino acid sequence encoded thereby, encoding a novel secreted matricellular proteins as disclosed herein that is related to SPARC.
  • SPARC is a prototypical matricellular protein known to be associated with various physiological processes such as the regulation of cell adhesion, cell migration, proliferation, counter adhesion, and growth factor expression and/or activity in numerous tissues.
  • hCRSPl polypeptide when used herein encompass native sequence hCRSPl polypeptide and polypeptide fragments and/or variants thereof (which are further defined herein) .
  • complementarity refers to the capacity of purine, pyrimidine, synthetic or modified nucleotides to associate by partial or complete complementarity through hydrogen or other bonding to form partial or complete double- or triple-stranded nucleic acid molecules.
  • the following base pairs occur by complete complementarity: (i) guanine (G) and cytosine (C) ; (ii) adenine (A) and thymine (T) ; and adenine (A) and uracil (U) .
  • Partial complementarity refers to association of two or more bases by one or more hydrogen bonds or attraction that is less than the complete complementarity as described above.
  • Partial or complete complementarity can occur between any two nucleotides, including naturally occurring or modified bases, e.g., as listed in 37 CFR ⁇ 1.822. All such nucleotides are included in polynucleotides of the invention as described herein.
  • Constant substitution or “conservative amino acid substitution” refers to a replacement of one or more amino acid residue (s) in a protein or peptide as stipulated in Table 1.
  • fusion protein denotes a hybrid protein molecule not found in nature comprising a translational fusion or enzymatic fusion in which two or more different proteins or fragments thereof are covalently linked on a single polypeptide chain.
  • polypeptide also includes such fusion proteins.
  • homolog or “homologous” describes the relationship between different nucleic acid molecules or amino acid sequences such that said sequences or molecules are related by partial identity or similarity at one or more regions within said molecules or sequences .
  • “Host cell” refers to any eucaryotic, procaryotic, or fusion or other cell or pseudo cell or membrane-containing construct that is suitable for propagating and/or expressing an isolated nucleic acid that is introduced into a host cell by any suitable means known in the art (e.g., but not limited to, transformation or transfection, or the like) , or induced to express an endogenous nucleic acid encoding an hCRSPl polypeptide according to the present invention.
  • the cell can be part of a tissue or organism, isolated in culture or in any other suitable form.
  • hybridization refers to a process in which a partially or completely single-stranded nucleic acid molecule joins with a complementary strand through nucleotide base pairing. Hybridization can occur under conditions of low, moderate or high stringency, with high stringency preferred. The degree of hybridization depends upon, for example, the degree of homology, the stringency conditions, and the length of hybridizing strands as known in the art.
  • isolated nucleic acid molecule is intended a nucleic acid molecule, DNA, RNA, or both which has been removed from its native or naturally occurring environment.
  • isolated nucleic acid molecules contained or generated in culture, a vector and/or a host cell are considered isolated for the purposes of the present invention.
  • isolated nucleic acid molecules include recombinant nucleic acid molecules maintained in heterologous host cells or purified (partially or substantially) nucleic acid molecules in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the nucleic acid molecules of the present invention.
  • Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically, purified from or provided in cells containing such nucleic acids, where the nucleic acid exists in other than a naturally occurring form, quantitatively or qualitatively .
  • isolated used in reference to at least one polypeptide of the invention describes a state of isolation such that the peptide or polypeptide is not in a naturally occurring form, and/or has been purified to remove at least some portion of cellular or non-cellular molecules with which the protein is naturally associated.
  • isolated may include the addition of other functional or structural polypeptides for a specific purpose, where the other peptide may occur naturally associated with at least one polypeptide of the present invention, but for which the resulting compound or composition does not exist naturally.
  • isolated used in reference to at least one antibody of the invention describes an antibody which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials, which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non- proteinaceous solutes.
  • the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue, or preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • mature protein or "mature polypeptide” as used herein refers to the form(s) of the protein produced by expression in a mammalian cell. It is generally hypothesized that once export of a growing protein chain across the rough endoplasmic reticulum has been initiated, proteins secreted by mammalian cells have a signal peptide (SP) sequence which is cleaved from the complete polypeptide to produce a "mature" form of the protein. Oftentimes, cleavage of a secreted protein is not uniform and may result in more than one species of mature protein. The cleavage site of a secreted protein is determined by the primary amino acid sequence of the complete protein and generally can not be predicted with complete accuracy.
  • SP signal peptide
  • cleavage point is after amino acid 20-30, preferably between 21-22, as presented in SEQ ID NO: 4.
  • the mature protein can be represented by, but not limited to, SEQ ID NO: 5.
  • the present invention provides polypeptides having a sequence of 90-100% of the contiguous sequence shown in SEQ ID NO: 4 which have an N-terminus beginning within 10 residues (i.e., + or - 10 residues) of the predicted cleavage point between amino acid 21 an22 of SEQ ID NO: 4.
  • cleavage sites for a secreted protein may be determined experimentally by amino- terminal sequencing of the one or more species of mature proteins found within a purified preparation of the protein.
  • a "nucleic acid probe, " "oligonucleotide probe, " or “probe” as used herein comprises at least one detectably labeled or unlabeled nucleic acid which hybridizes under specified hybridization conditions with at least one other nucleic acid.
  • a nucleic acid probe may be an oligonucleotide or a nucleotide polymer.
  • a probe can optionally contain a detectable moiety which may be attached to the end(s) of the probe or be internal to the sequence of the probe, termed a "detectable probe” or “detectable nucleic acid probe.”
  • plasmid refers to an extrachromosomal genetic element.
  • the plasmids disclosed herein are commercially available, publicly available on an unrestricted basis, or can be constructed from readily available plasmids in accordance with published procedures.
  • a "polynucleotide” comprises at least 10-20 nucleotides of a nucleic acid (RNA, DNA or combination thereof) , provided by any means, such as synthetic, recombinant isolation or purification method steps.
  • a “primer” is a nucleic acid fragment or oligonucleotide which functions as an initiating substrate for enzymatic or synthetic elongation of, for example, a nucleic acid molecule, e.g., using an amplification reaction, such as, but not limited to, a polymerase chain reaction (PCR) , as known in the art.
  • PCR polymerase chain reaction
  • promoter refers to a nucleic acid sequence that directs transcription, for example, of DNA to RNA.
  • An inducible promoter is one that is regulatable by environmental signals, such as carbon source, heat, or metal ions, for example.
  • a constitutive promoter generally operates at a constant level and is not regulatable.
  • Recombinant DNA cloning vector refers to any autonomously replicating agent, including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been incorporated.
  • expression vector refers to any recombinant DNA cloning vector, for example a plasmid or phage, in which a promoter and other regulatory elements are present thereby enabling transcription of an inserted DNA, which may encode a protein.
  • stringency refers to hybridization conditions for nucleic acids in solution. High stringency conditions disfavor non-homologous base pairing. Low stringency conditions have much less of this effect. Stringency may be altered, for example, by changes in temperature and/or salt concentration, or other conditions, as well known in the art.
  • a non-limiting example of "high stringency” conditions includes, for example, (a) a temperature of about 42°C , a formamide concentration of about 20%, and a low salt (SSC) concentration, or, alternatively, a temperature of about 65°C, or less, and a low salt (SSPE) concentration; (b) hybridization in 0.5 M NaHP04 , 7% sodium dodecyl sulfate (SDS), 1 M EDTA at 65°C (See, e.g., Ausubel, et al . , ed. , Current Protocols in Molecular Biology, 1987-1998, Wiley Interscience, New York, at ⁇ 2.10.3).
  • SSC comprises a hybridization and wash solution.
  • a stock 20X SSC solution contains 3M sodium chloride, 0.3M sodium citrate, pH 7.0.
  • SSPE comprises a hybridization and wash solution.
  • a IX SSPE solution contains 180 mM NaCl, 9mM Na2HP04 , 0.9 mM NaH2P04 and 1 mM EDTA, pH 7.4.
  • treatment describes the management and care of a mammal for the purpose of combating a disease, condition, or disorder and includes the administration of a hCRSPl agonist, hCRSPl antagonist, hCRSPl polynucleotide, hCRSPl polypeptide, hCRSPl variant, and/or hCRSPl antibody to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder.
  • the disease, condition, or disorder to be treated includes, but is not limited to, one or more of the following disorders: nerve damage, neurodegeneration, trigeminal neuralgia, glossopharyngeal neuralgia, Bell's Palsy, myasthenia gravis, muscular dystrophy, progressive muscular atrophy, progressive bulbar inherited muscular atrophy, herniated, ruptured or prolapsed invertebrae disk, cervical spondylosis, plexus disorders, thoracic outlet destruction, peripheral neuropathy, such as those caused by lead, dapsone, ticks or porphyria, peripheral myelin disorders, Alzheimer's disease, Gullain-Barre syndrome, Parkinson's disease, Parkinsonian disorders, ALS, multiple sclerosis, other central myelin disorders, stroke, ischemia associated with stroke, neural paropathy, other neural degenerative diseases, motor neuron diseases, sciatic crush, neuropathy associated with diabetes, spinal cord injuries, facial nerve crush, chemotherapy- or pharma
  • hCRSPl polynucleotide hCRSPl polypeptide, or hCRSPl antibody and/or any fragment thereof may result in functional variants that display equivalent or superior functional characteristics when compared to the hCRSPl polynucleotides , polypeptides, and antibodies of the present invention.
  • the present invention further contemplates alterations in the hCRSPl polynucleotides, polypeptides, antibodies and/or fragments thereof that may include one or more nucleotide or amino acid insertions, deletions, substitutions, truncations, fusions, shuffling of subunit sequences, and the like, either from natural mutations or human manipulation, provided that the sequences produced by such modifications have substantially the same (or improved or reduced, as may be desirable) activity (ies) as the hCRSPl polynucleotide, polypeptide, antibody, and/or fragment thereof and/or variant thereof disclosed herein.
  • variant refers to a polynucleotide as shown in SEQ ID NO : 1 , or 3 , or any fragment thereof, a polypeptide as shown in SEQ ID NO: 4 or 5, as well any fragment thereof, or a hCRSPl antibody that further comprises at least one of the various types of modifications contemplated herein.
  • variant as applied to a hCRSPl polypeptide of the present invention, is intended to refer to a "functional" hCRSPl polypeptide, as defined herein, having at least about 90% amino acid sequence identity with a sequence of amino acids shown in SEQ ID NO : 4 or 5.
  • hCRSPl polypeptide variants include, for instance, hCRSPl polypeptides or fragments thereof wherein one or more amino acid residues are added, substituted or deleted, at the N- or C-terminus or within the sequence of SEQ ID NO:4 or 5.
  • a hCRSPl variant will have at least about 90% amino acid sequence identity, preferably at least about 91% sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity, yet more preferably at least about 99% amino acid sequence identity with the amino acid sequence as shown in SEQ ID NO: 4 or 5, or any fragment thereof.
  • variant in reference to a hCRSPl polynucleotide is intended to refer to a nucleic acid molecule having at least about 70% nucleic acid sequence identity with a polynucleotide sequence shown in SEQ ID NO:l, 2, or 3 , or any fragment thereof.
  • an hCRSPl polynucleotide variant will have at least about 70% nucleic acid sequence identity, more preferably at least about 80% nucleic acid sequence identity, yet more preferably at least about 81% nucleic acid sequence identity, yet more preferably at least about 82% nucleic acid sequence identity, yet more preferably at least about 83% nucleic acid sequence identity, yet more preferably at least about 84% nucleic acid sequence identity, yet more preferably at least about 85% nucleic acid sequence identity, yet more preferably at least about 86% nucleic acid sequence identity, yet more preferably at least about 87% nucleic acid sequence identity, yet more preferably at least about 88% nucleic acid sequence identity, yet more preferably at least about 89% nucleic acid sequence identity, yet more preferably at least about 90% nucleic acid sequence identity, yet more preferably at least about 91% nucleic acid sequence identity, yet more preferably at least about 92% nucleic acid sequence identity, yet more preferably at least about 93% nucleic acid sequence identity, yet more
  • Percent (%) nucleic acid sequence identity with respect to the hCRSPl sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the hCRSPl sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as ALIGN, Align-2, Megalign (DNASTAR) , or BLAST (e.g., Blast, Blast-2) software.
  • a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the polypeptide-encoding nucleic acid molecule of interest and the comparison nucleic acid molecule of interest (i.e., the sequence against which the polypeptide-encoding nucleic acid molecule of interest is being compared) as determined by WU-BLAST-2 by (b) the total number of nucleotides of the hCRSPl polypeptide-encoding nucleic acid molecule of interest.
  • Percent (%) amino acid sequence identity with respect to the hCRSPl amino acid sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in an hCRSPl polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as ALIGN, ALIGN-2, Megalign (DNASTAR) or BLAST (e.g., Blast, Blast-2, WU-Blast- 2) software.
  • a % amino acid sequence identity value is determined by divided (a) the number of matching identical ammo acid residues between the amino acid sequence of the hCRSPl polypeptide of interest and the comparison amino acid sequence of interest (i.e., the sequence against which the hCRSPl polypeptide of interest is being compared) as determined by WU-BLAST-2 by (b) the total number of amino acid residues of the hCRSPl polypeptide of interest, respectively.
  • vector refers to a nucleic acid compound used for introducing exogenous or endogenous nucleic acid into host cells.
  • a vector comprises a nucleotide sequence which may encode one or more polypeptide molecules. Plasmids, cosmids, viruses and bacteriophages, in a natural state or which have undergone recombinant engineering, are non-limiting examples of commonly used vectors to provide recombinant vectors comprising at least one desired isolated nucleic acid molecule.
  • nucleic acid molecule of the present invention encoding an hCRSPl polypeptide can be obtained using well-known methods.
  • Nucleic acid molecules of the present invention can be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including, but not limited to, cDNA and genomic DNA obtained by cloning or produced synthetically, or any combination thereof.
  • the DNA can be triple-stranded, double-stranded or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also referred to as the anti-sense strand.
  • Isolated nucleic acid molecules of the present invention include nucleic acid molecules comprising an open reading frame (ORF) shown in at least one of SEQ ID NOS:l, 2, 3; nucleic acid molecules comprising the coding sequence for an hCRSPl polypeptide; and nucleic acid molecules which comprise a nucleotide sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode at least one hCRSPl polypeptide as described herein.
  • ORF open reading frame
  • nucleic acid molecules comprising the coding sequence for an hCRSPl polypeptide
  • the genetic code is well known in the art.
  • the isolated nucleic acid comprises DNA having at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91% sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity, yet more preferably at least about 99% sequence identity to (a) a DNA molecule en
  • the invention concerns an isolated nucleic acid molecule encoding an hCRSPl polypeptide comprising DNA hybridizing to the complement of the nucleic acid between about nucleotides: (a) 1 to about 1338, inclusive, of SEQ ID NO : 2 and (b ) 1 to about 1275, inclusive, of SEQ ID NO: 3.
  • the invention concerns an isolated nucleic acid molecule encoding an active hCRSPl polypeptide comprising a nucleotide sequence that hybridizes to the complement of a nucleic acid sequence that encodes amino acids (a) 1 or about 10 to about 21, inclusive, SEQ ID N0:4 or (b) 1 or about 22 to about 446, inclusive, of SEQ ID NO : 4.
  • hybridization occurs under stringent hybridization and wash conditions.
  • the invention concerns an isolated nucleic acid molecule produced by hybridizing a test DNA molecule under stringent conditions with: (a) a DNA molecule encoding (i) an hCRSPl polypeptide having the sequence of amino acid residues from about 1 or about 22 to about 446, inclusive, of SEQ ID NO :4, or (b) the complement of the DNA molecule of (a) , and if the DNA molecule has at least about an 80% sequence identity, preferably at least about an 81% sequence identity, more preferably at least about a 82% sequence identity, yet more preferably at least about a 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91% sequence identity, yet more preferably at least about 90%
  • the invention concerns an isolated nucleic acid molecule comprising: (a) DNA encoding a polypeptide scoring at least about 80% positives, preferably at least about 81% positives, more preferably at least about 82% positives, yet more preferably at least about 83% positives, yet more preferably at least about 84% positives, yet more preferably at least about 85% positives, yet more preferably at least about 86% positives, yet more preferably at least about 87% positives, yet more preferably at least about 88% positives, yet more preferably at least about 89% positives, yet more preferably at least about 90% positives, yet more preferably at least about 91% positives, yet more preferably at least about 92% positives, yet more preferably at least about 93% positives, yet more preferably at least about 94% positives, yet more preferably at least about 95% positives, yet more preferably at least about 96% positives, yet more preferably at least about 97% positives, yet more preferably at least about 98% positives, yet
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding an hCRSPl polypeptide without the N-terminal signal sequence and/or initiating methionine, or is complementary to such encoding nucleic acid molecule.
  • the signal peptide has been tentatively identified as extending from about amino acid residue 1 to about amino acid residue 21, inclusive, in the sequence of SEQ ID NO: 4.
  • the invention also provides an isolated nucleic acid molecule having the nucleotide sequence shown in at least one of SEQ ID N0S:1, 2, 3, or a nucleic acid molecule having a sequence complementary thereto.
  • isolated molecules particularly nucleic acid molecules, are useful as probes for gene mapping by in situ hybridization with chromosomes, and for detecting transcription, translation and/or expression of the hCRSPl gene in human tissue, for instance, by Northern blot analysis for mRNA detection.
  • nucleotide sequences identified by sequencing a nucleic acid molecule herein can be or were identified using an automated nucleic acid sequencer, and all amino acid sequences of polypeptides encoded by nucleic acid molecules identified herein can be or were identified by codon correspondence or by translation of a nucleic acid sequence identified using method steps as described herein or as known in the art. Therefore, as is well known in the art that for any nucleic acid sequence identified by this automated approach, any nucleotide sequence identified herein may contain some errors which are reproducibly correctable by re-sequencing using well-known methods.
  • Nucleotide sequences identified by automation are typically at least about 95% to at least about 99.999% identical to the actual nucleotide sequence of the sequenced nucleic acid molecule.
  • the actual sequence can be more precisely identified by other approaches including manual nucleic acid sequencing methods well known in the art.
  • a single insertion or deletion in an identified nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the identified amino acid sequence encoded by an identified nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced nucleic acid molecule, beginning at the point of such an insertion or deletion.
  • the present invention is further directed to fragments of a hCRSPl-encoding polynucleotide sequence that may find use as, for example, hybridization probes or for encoding fragments of an hCRSPl polypeptide that may optionally encode a polypeptide comprising a binding site for an anti- hCRSPl antibodyusing the methods disclosed herein.
  • Other useful fragments of the hCRSPl nucleic acids include antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target hCRSPl mRNA (sense) of hCRSPl DNA (anti-sense) sequences.
  • Antisense or sense oligonucleotides comprise a fragment of the coding region of hCRSPl DNA. Such a fragment generally comprises at least about 14 nucleotides. preferably from about 14 to 30 nucleotides.
  • the ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example. Stein and Cohen, Cancer Rex ⁇ 2659 (1988) and van der Krol et al.. BioTechniques 6: 958 (1988).
  • binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block transcription or translation of the target sequence by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means.
  • the antisense oligonucleotides thus may be used to block expression of hCRSPl proteins.
  • Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO 91106629) and wherein such sugar linkages are resistant to endogenous nucleases.
  • Such oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences.
  • sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10048, and other moieties that increase affinity of the oligonucleotide for a target nucleic acid sequence, such poly-L-lysinc .
  • intercalating agents such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.
  • Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaP0 4 mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus.
  • an antisense or sense oligonucleotide is inserted into a suitable retroviral vector.
  • a cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector, either in vivo or ex vivo.
  • Suitable retroviral vectors include, but are not limited to, those derived from the murine retrovirus M-MSV, N2 (a retrovirus derived from M-MuLV) , or the double copy vectors designated CDTSA, CTSB and DCTSC (see WO 90/13641) .
  • Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753.
  • Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other iigands that bind to cell surface receptors.
  • conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
  • a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448.
  • the sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.
  • a fragment of an isolated nucleic acid molecule is meant a molecule having at least 10 nucleotides of a nucleotide sequence of a deposited cDNA or a nucleotide sequence shown in at least one of SEQ ID NOS : 1 , 2, 3, and is intended to mean fragments at least about 10 nucleotides, which are useful, inter alia as diagnostic probes and primers as described herein.
  • fragments which include 10 or more contiguous nucleotides from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NOS:l, 2, 3, as determined by methods known in the art (See e.g., Ausubel, supra, Chapter 7) .
  • fragments such as at least about 50, 100, 120, 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, and/or 4000 or more nucleotides in length, are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence as shown at least one of SEQ ID NOS:l, 2 , 3 , or any previously referenced deposited cDNA.
  • nucleic acids fragments are usually at least about 20 nucleotides in length, preferably at least about 30 nucleotides in length, more preferable at least about 40 nucleotides in length, yet more preferably at least about 50 nucleotides in length, yet more preferably at least about 60 nucleotides in length, yet more preferably at least about 70 nucleotides in length, yet more preferably at least about 50 nucleotides in length, yet more preferably at least about 90 nucleotides in length, yet more preferably at least about 100 nucleotides in length, yet more preferably at least about 110 nucleotides in length, yet more preferably at least about 120 nucleotides in length, yet more preferably at least about 130 nucleotides in length, yet more preferably at least about 140 nucleotides in length, yet more preferably at least about 150 nucleotides in length, yet more preferably at least about 160 nucleotides in length, yet more preferably at least about 170 nucle
  • nucleotide fragments are also useful according to the present invention for screening DNA, cDNA, or mRNA sequences that code for one or more fragments of an hCRSPl polypeptide as described herein.
  • screening as a non- limiting example can include the use of so-called "DNA chips" for screening DNA, cDNA, or mRNA sequences of the present invention, as described, e.g., in U.S. Patent Nos. 5,631,734, 5,624,711, 5,744,305, 5,770,456, 5,770,722, 5,675,443, 5,695,940, 5,710,000, 5,733,729, which are entirely incorporated herein by reference.
  • a polynucleotide which hybridizes only to a poly A sequence such as the 3' terminal poly (A) of an hCRSPl cDNA shown in at least one of SEQ ID NOS:l, 2, 3, or to a complementary stretch of T (or U) resides, would not be included in a probe of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
  • the present invention also provides subsequences of full-length nucleic acids. Any number of subsequences can be obtained by reference to at least one of SEQ ID NOS:l, 2, 3, or a complementary sequence, and using primers which selectively amplify, under stringent conditions to: at least two sites to the polynucleotides of the present invention, or to two sites within the nucleic acid which flank and comprise a polynucleotide of the present invention, or to a site within a polynucleotide of the present invention and a site within the nucleic acid which comprises it.
  • a variety of methods for obtaining 5' and/or 3' ends is well known in the art.
  • the present invention provides hCRSPl polynucleotides having the sequence of the hCRSPl gene, nuclear transcript, cDNA, or complementary sequences and/or subsequences thereof.
  • Primer sequences can be obtained by reference to a contiguous subsequence of a polynucleotide of the present invention.
  • Primers are chosen to selectively hybridize, under PCR amplification conditions, to a polynucleotide of the present invention in an amplification mixture comprising a genomic and/or cDNA library from the same species.
  • the primers are complementary to a subsequence of the amplified nucleic acid.
  • the primers will be constructed to anneal at their 5' terminal ends to the codon encoding the carboxy or amino terminal amino acid residue (or the complements thereof) of the polynucleotides of the present invention.
  • the primer length in nucleotides is selected from the group of integers consisting of from at least 15 to 50.
  • the primers can be at least 15, 18, 20, 25, 30, 40, or 50 nucleotides in length or any range or value therein.
  • a non-annealing sequence at the 5' end of the primer (a "tail") can be added, for example, to introduce a cloning site at the terminal ends of the amplified DNA.
  • the amplification primers may optionally be elongated in the 3 ' direction with additional contiguous or complementary nucleotides from the polynucleotide sequences, such as shown in at least one of SEQ ID NOS:l, 2, 3, from which they are derived.
  • the number of nucleotides by which the primers can be elongated is selected from the group of integers consisting of from at least 1 to at least 25.
  • the primers can be elongated with an additional 1, 5, 10, or 15 nucleotides or any range or value therein.
  • a lengthened primer sequence can be employed to increase specificity of binding (i.e., annealing) to a target sequence, or to add useful sequences, such as links or restriction sites (See e.g., Ausubel, supra, Chapter 15).
  • the amplification products can be translated using expression systems well known to those of skill in the art and as discussed, infra.
  • the resulting translation products can be confirmed as polypeptides of the present invention by, for example, assaying for the appropriate catalytic activity (e.g., specific activity and/or substrate specificity), or verifying the presence of one or more linear epitopes which are specific to a polypeptide of the present invention.
  • the present invention provides isolated nucleic acids that hybridize under selective hybridization conditions to a polynucleotide disclosed herein, e.g., SEQ ID NO:l.
  • a polynucleotide disclosed herein e.g., SEQ ID NO:l.
  • the polynucleotides of this embodiment can be used for isolating, detecting, and/or quantifying nucleic acids comprising such polynucleotides.
  • polynucleotides of the present invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library.
  • the polynucleotides are genomic or cDNA sequences isolated, or otherwise complementary to, a cDNA from a human or mammalian nucleic acid library.
  • the cDNA library comprises at least 80% full-length sequences, preferably at least 85% or 90% full- length sequences, and more preferably at least 95% full- length sequences.
  • the cDNA libraries can be normalized to increase the representation of rare sequences.
  • Low stringency hybridization conditions are typically, but not exclusively, employed with sequences having a reduced sequence identity relative to complementary sequences.
  • Moderate and high stringency conditions can optionally be employed for sequences of greater identity.
  • Low stringency conditions allow selective hybridization of sequences having about 70% sequence identity and can be employed to identify orthologous or paralogous sequences .
  • polynucleotides of this invention will encode an epitope of a polypeptide described herein.
  • the polynucleotides of this invention embrace nucleic acid sequences that can be employed for selective hybridization to a polynucleotide encoding a polypeptide of the present invention.
  • Screening polypeptides for specific binding to antibodies or fragments can be conveniently achieved using peptide display libraries. This method involves the screening of large collections of peptides for individual members having the desired function or structure.
  • Antibody screening of peptide display libraries is well known in the art.
  • the displayed peptide sequences can be from 3 to 5000 or more amino acids in length, frequently from 5-100 amino acids long, and often from about 8 to 15 amino acids long.
  • the present invention provides isolated nucleic acids comprising hCRSPl polynucleotides, wherein the polynucleotides are complementary to the polynucleotides described herein.
  • complementary sequences base pair throughout the entirety of their length with such polynucleotides (i.e., have 100% sequence identity over their entire length) .
  • Complementary bases associate through hydrogen bonding in double-stranded nucleic acids.
  • the following base pairs are complementary: guanine and cytosine; adenine and thymine; and adenine and uracil (See, e.g., Ausubel, supra, Chapter 67; or Sambrook, supra) .
  • the isolated nucleic acids of the present invention can be made using (a) standard recombinant methods, (b) synthetic techniques, (c) purification techniques, or combinations thereof, as well known in the art.
  • the nucleic acids may conveniently comprise sequences in addition to a polynucleotide of the present invention.
  • a multi-cloning site comprising one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in isolation of the polynucleotide.
  • translatable sequences may be inserted to aid in the isolation of the translated polynucleotide of the present invention.
  • a hexa-histidine marker sequence provides a convenient means to purify the proteins of the present invention.
  • the nucleic acid of the present invention - excluding the polynucleotide sequence - is optionally a vector, adapter, or linker for cloning and/or expression of a polynucleotide of the present invention. Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell.
  • the length of a nucleic acid of the present invention less the length of its polynucleotide of the present invention is less than 20 kilobase pairs, often less than 15 kb, and frequently less than 10 kb.
  • Use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra, Chapters 1-5; or Sambrook, supra)
  • RNA, cDNA, genomic DNA, or a hybrid thereof can be obtained from biological sources using any number of cloning methodologies known to those of skill in the art.
  • oligonucleotide probes that selectively hybridize, under stringent conditions, to the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library. While isolation of RNA, and construction of cDNA and genomic libraries is well known to those of ordinary skill in the art. (See, e.g., Ausubel, supra, Chapters 1-7; or Sambrook, supra)
  • a cDNA or genomic library can be screened using a probe based upon the sequence of a polynucleotide of the present invention, such as those disclosed herein. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms.
  • Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms.
  • degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent . As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for duplex formation to occur. Temperature, ionic strength, pH and the presence of a partially denaturing solvent such as forma ide can control the degree of stringency.
  • RNA or DNA RNA or DNA are well known in the art and can be used according to the present invention without undue experimentation, based on the teaching and guidance presented herein.
  • RNA amplification processes include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (see, e.g., U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188, to Mullis, et al.; 4,795,699 and 4,921,794 to Tabor, et al ; 5,142,033 to Innis; 5,122,464 to Wilson, et al .
  • PCR polymerase chain reaction
  • RNA mediated amplification which uses anti- sense RNA to the target sequence as a template for double- stranded DNA synthesis (U.S. Patent No. 5,130,238 to Malek, et al, with the tradename NASBA) , the entire contents of which are herein incorporated by reference. (See, e.g., Ausubel, supra, Chapter 15; or Sambrook, supra)
  • PCR polymerase chain reaction
  • in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes.
  • examples of techniques sufficient to direct persons of skill through in vi tro amplification methods are found in Berger, Sambrook, and Ausubel (e.g., Chapter 15) supra, as well as Mullis, et al . , U.S. Patent No.
  • the isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis by methods such as the phosphotriester method of Narang, et al . , Meth. Enzymol. 68:90-99 (1979); the phosphodiester method of Brown, et al., Meth. Enzymol. 68:109-151 (1979); the diethyhCRSPlhosphoramidite method of Beaucage, et al . , Tetra . Letts. 22:1859-1862 (1981); the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetra. Letts.
  • Chemical synthesis generally produces a single-stranded oligonucleotide, which may be converted into double-stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template.
  • an automated synthesizer e.g., as described in Needham-VanDevanter, et al., Nucleic Acids Res. 12:6159-6168 (1984); and the solid support method of U.S. Patent No. 4,458,066.
  • Chemical synthesis generally produces a single-stranded oligonucleotide, which may be converted into double-stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template.
  • One of skill in the art will recognize that while chemical synthesis of DNA can be limited to sequences of about 100 or more bases, longer sequences may be obtained by the ligation of shorter sequences.
  • the present invention further provides recombinant expression cassettes comprising a nucleic acid of the present invention.
  • a nucleic acid sequence of the present invention for example, a cDNA or a genomic sequence encoding a full- length polypeptide of the present invention, can be used to construct a recombinant expression cassette which can be introduced into at least one desired host cell.
  • a recombinant expression cassette will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences that will direct the transcription of the polynucleotide in the intended host cell .
  • heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the present invention. These promoters can also be used, for example, in recombinant expression cassettes to drive expression of antisense nucleic acids to reduce, increase, or alter hCRSPl content and/or composition in a desired tissue.
  • isolated nucleic acids which serve as promoter or enhancer elements can be introduced in the appropriate position (generally upstream) of a non- heterologous form of a polynucleotide of the present invention so as to up or down regulate expression of a polynucleotide of the present invention.
  • endogenous promoters can be altered in vivo or in vi tro by mutation, deletion and/or substitution.
  • a polynucleotide of the present invention can be expressed in either sense or anti-sense orientation as desired. It will be appreciated that control of gene expression in either sense or anti-sense orientation can have a direct impact on the observable characteristics.
  • Another method of suppression is sense suppression.
  • Introduction of nucleic acid configured in the sense orientation has been shown to be an effective means by which to block the transcription of target genes .
  • cross-linking agents, alkylating agents and radical generating species as pendant groups on polynucleotides of the present invention can be used to bind, label, detect and/or cleave nucleic acids.
  • the present invention also relates to vectors that include isolated nucleic acid molecules of the present invention, host cells that are genetically engineered with the recombinant vectors, and the production of hCRSPl polypeptides or fragments thereof by recombinant techniques, as is well known in the art. See, e.g., Sambrook, et al., supra ; Ausubel, supra, Chapters 1-9, each entirely incorporated herein by reference.
  • the polynucleotides can optionally be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, or any other suitable promoter.
  • an appropriate promoter such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, or any other suitable promoter.
  • the skilled artisan will know other suitable promoters.
  • the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome- binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNA to be translated, with VAA and VAG preferred for mammalian or eukaryotic cell expression.
  • a translation initiating at the beginning e.g., UAA, UGA or UAG
  • a termination codon e.g., UAA, UGA or UAG
  • Expression vectors will preferably include at least one selectable marker.
  • markers include, e.g., dihydrofolate reductase, ampicillin (G418) , hygromycin or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria or prokaryotic ⁇ .
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli , Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells.
  • Vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • Preferred eucaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3 , pBPV, pMSG and pSVL available from Pharmacia.
  • Other suitable vectors will be readily apparent to the skilled artisan. See, e.g., Ausubel, supra , Chapter 1; Coligan, Current Protocols in Protein Science, supra , Chapter 5.
  • Introduction of a vector construct into a host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Sambrook, supra , Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, 16.
  • nucleic acid molecules of the present invention which comprise a nucleic acid encoding an hCRSPl polypeptide can include, but are not limited to, those encoding the amino acid sequence of the mature polypeptide; the coding sequence for the mature polypeptide and additional sequences, such as the coding sequence of at least one signal leader or fusion peptide; the mature polypeptide, with at least one intron, together with additional, non-coding sequences such as, but not limited to, introns and non-coding 5' and 3' sequences (i.e., the transcribed, non-translated sequences that play a role in transcription, ribosome binding, stability, or mRNA processing, including splicing and polyadenylation signals) .
  • any of the above described nucleic acids can further comprise additional coding sequences which encodes additional amino acids which provide additional functionalities known to one skilled in the art to be useful additions to such nucleic acids.
  • the sequence encoding a polypeptide can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused polypeptide.
  • Preferred nucleic acid fragments of the present invention also include nucleic acid molecules encoding epitope-bearing portions of an hCRSPl polypeptide.
  • Polypeptide (s) of the present invention can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of a polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to a polypeptide to facilitate purification. Such regions can be removed prior to final preparation of a polypeptide. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17 and 18; Ausubel, supra, Chapters 16, 17 and 18. Expression of Proteins in Host Cells
  • nucleic acids of the present invention may express a protein of the present invention in a recombinantly engineered cell, such as bacteria, yeast, insect, or mammalian cells.
  • the cells produce the protein in a non- natural condition (e.g., in quantity, composition, location, and/or time) , because they have been genetically altered through human intervention to do so .
  • a non- natural condition e.g., in quantity, composition, location, and/or time
  • the expression of isolated nucleic acids encoding a protein of the present invention will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter (which is either constitutive or inducible) followed by incorporation into an expression vector.
  • the vectors can be suitable for replication and integration in either prokaryotes or eukaryotes.
  • Typical expression vectors contain transcription and translation terminators, initiation sequences and promoters useful for regulation of the expression of the DNA encoding a protein of the present invention.
  • expression vectors which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator.
  • a strong promoter to direct transcription
  • a ribosome binding site for translational initiation to translational initiation
  • a transcription/translation terminator to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein.
  • modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly
  • nucleic acids of the present invention can be expressed in a host cell by turning on (by manipulation) in a host cell that contains endogenous DNA encoding a polypeptide of the present invention.
  • Such methods are well known in the art, e.g., as described in US patent Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirely incorporated herein by reference.
  • Prokaryotic cells may be used as hosts for expression. Prokaryotes most frequently are represented by various strains of E. coli; however, other microbial strains may also be used. Commonly used prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta lactamase (penicillinase) and lactose (lac) promoter systems (Chang, et al . , Nature 198:1056 (1977)), the tryptophan (trp) promoter system (Goeddel, et al . , Nucleic Acids Res.
  • DNA vectors transfected in E. coli are also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol .
  • the vector is selected to allow introduction into the appropriate host cell .
  • Bacterial vectors are typically of plasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If a plasmid vector is used, the bacterial cells are transformed with the plasmid vector DNA.
  • Expression systems for expressing a protein of the present invention are available using Bacillus sp. and Salmonella (Palva, et al . , Gene 22:229-235 (1983); Mosbach, et al., Nature 302:543-545 (1983)). See, e.g., Ausubel, supra, Chapters 1-3, 16 (Sec.1); and Coligan, supra, Current Protocols in Protein Science, Units 5.1, 6.1-6.7.
  • eukaryotic expression systems such as yeast, insect cell lines, plant and mammalian cells
  • yeast eukaryotic expression systems
  • a nucleic acid of the present invention can be expressed in these eukaryotic systems.
  • Synthesis of heterologous proteins in yeast is well known.
  • F. Sherman, et al . Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982) is a well-recognized work describing the various methods available to produce the protein in yeast.
  • Two widely utilized yeast for production of eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris.
  • Vectors, strains, and protocols for expression in Saccharomyces and Pichia are known in the art and available from commercial suppliers (e.g., Invitrogen) . Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences and the like as desired.
  • a protein of the present invention once expressed, can be isolated from yeast by lysing the cells and applying standard protein isolation techniques to the lysates . The monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay of other standard immunoassay techniques.
  • sequences encoding proteins of the present invention can also be ligated to various expression vectors for use in transfecting cell cultures of, for instance, mammalian, insect, or plant origin.
  • Illustrative of cell cultures useful for the production of the peptides are mammalian cells. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used.
  • a number of suitable host cell lines capable of expressing intact proteins have been developed in the art, and include the HEK293, BHK21, and CHO cell lines.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter) , an enhancer (Queen, et al . , Immunol. Rev. 89:49 (1986)), and processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences.
  • Other animal cells useful for production of proteins of the present invention are available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (7th edition, 1992) .
  • Appropriate vectors for expressing proteins of the present invention in insect cells are usually derived from the SF9 baculovirus.
  • suitable insect cell lines include mosquito larvae, silkworm, armyworm, moth and Drosophila cell lines such as a Schneider cell line (See Schneider, J. Embryol. Exp. Morphol. 27:353-365 (1987).
  • yeast when higher animal or plant host cells are employed, polyadenlyation or transcription terminator sequences are typically incorporated into the vector.
  • An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included.
  • splicing sequence is the VP1 intron from SV40 (Sprague, et al . , J. Virol. 45:773-781 (1983)).
  • gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type-vectors. M. Saveria- Campo, bovine Papilloma Virus DNA, a Eukaryotic Cloning Vector in DNA Cloning Vol. II, a Practical Approach, D. M. Glover, Ed., IRL Press, Arlington, VA, pp. 213-238 (1985).
  • An hCRSPl polypeptide can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.
  • Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eucaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells.
  • polypeptides of the present invention can be glycosylated or can be non- glycosylated.
  • polypeptides of the invention can also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20.
  • the invention further provides an isolated hCRSPl polypeptides having fragments or specified variants of the amino acid sequence encoded by the deposited cDNAs, or the amino acid sequence in SEQ ID NO: 4 or 5.
  • the isolated proteins of the present invention comprise a polypeptide encoded by any one of the polynucleotides of the present invention as discussed herein, or polypeptides which are specified fragments or variants thereof.
  • Exemplary polypeptide sequences are provided in SEQ ID NO : 4 or 5.
  • the proteins of the present invention or variants thereof can comprise any number of contiguous amino acid residues from a polypeptide of the present invention.
  • the subsequence of contiguous amino acids is at least 20, 30, 40, 50, 60, 70, 80, or 90 amino acids in length.
  • the number of contiguous amino acid residues in such subsequences can be any integer selected from the group consisting of from 1 to 20, such as 2 , 3, 4, or 5.
  • the invention concerns an isolated hCRSPl polypeptide comprising an amino acid sequence having at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, yet more preferably at least about 83% sequence identity, yet more preferably at least about 84% sequence identity, yet more preferably at least about 85% sequence identity, yet more preferably at least about 86% sequence identity, yet more preferably at least about 87% sequence identity, yet more preferably at least about 88% sequence identity, yet more preferably at least about 89% sequence identity, yet more preferably at least about 90% sequence identity, yet more preferably at least about 91% sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity, yet more preferably at least about 99%
  • the invention concerns an isolated hCRSPl polypeptide comprising an amino acid sequence scoring at least about 80% positives, preferably at least about 81% positives, more preferably at least about 82% positives, yet more preferably at least about 83% positive, yet more preferably at least about 84% positives, yet more preferably at least about 85% positives, yet more preferably at least about 86% positives, yet more preferably at least about 87% positives, yet more preferably at least about 88% positives, yet more preferably at least about 89% positives, yet more preferably at least about 90% positives, yet more preferably at least about 91% positives, yet more preferably at least about 92% positives, yet more preferably at least about 93% positives, yet more preferably at least about 94% positives, yet more preferably at least about 95% positives, yet more preferably at least about 96% positives, yet more preferably at least about 97% positives, yet more preferably at least about 98% positives, yet more preferably at least about 99% positive
  • the invention provides an isolated hCRSPl polypeptide without the N-terminal signal sequence and/or initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore described.
  • Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of hCRSPl polypeptide and recovering the hCRSPl polypeptide, respectively, from the cell culture.
  • the invention provides a polypeptide produced by: (1) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a (i) hCRSPl polypeptide having the sequence of amino acid residues from about 22 to about 446, inclusive, of SEQ ID NO:
  • test DNA molecule has at least about an 80% sequence identity, preferably at least about an 81% sequence identity, more preferably at least about an 82% sequence identity, yet more preferably at least about an 83% sequence identity, yet more preferably at least about an 84% sequence identity, yet more preferably at least about an 85% sequence identity, yet more preferably at least about an 86% sequence identity, yet more preferably at least about an 87% sequence identity, yet more preferably at least about an 88% sequence identity, yet more preferably at least about an 89% sequence identity, yet more preferably at least about a 90% sequence identity, yet more preferably at least about a 91% sequence identity, yet more preferably at least about a 92% sequence identity, yet more preferably at least about a 93% sequence identity, yet more preferably at least about a 94% sequence identity, yet more preferably at least about a 95% sequence identity, yet more preferably at least about a 96% sequence identity, yet more preferably at least about preferably at least about preferably at least about
  • the invention concerns an isolated hCRSPl polypeptide comprising the sequence of amino acid residues from about 1 or about 22 to about 446, inclusive, of SEQ ID NO : 4, or a fragment or variant thereof which is biologically active or sufficient to provide a binding site for an anti-hCRSPl antibody, wherein the identification of hCRSPl polypeptide or fragments thereof that possess biological activity or provide a binding site for an anti-hCRSPl antibody may be accomplished in a routine manner using techniques which are well known in the art.
  • the present invention includes biologically active polypeptides of the present invention (i.e., enzymes).
  • biologically active polypeptides have a specific activity at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95%-1000% of that of the native (non- synthetic) , endogenous polypeptide.
  • the substrate specificity e.g., k cat /Km
  • the K m will be at least 30%, 40%, or 50%, that of the native (non-synthetic), endogenous polypeptide; and more preferably at least 60%, 70%, 80%, or 90%-1000%.
  • Methods of assaying and quantifying measures of enzymatic activity and substrate specificity are well known to those of skill in the art.
  • polypeptides of the present invention will, when presented as an immunogen, elicit production of an antibody specifically reactive to a polypeptide of the present invention encoded by a polynucleotide of the present invention as described, supra.
  • Exemplary polypeptides include those which are full-length, such as those disclosed herein.
  • the proteins of the present invention will not bind to antisera raised against a polypeptide of the present invention which has been fully immunosor/bed with the same polypeptide.
  • Immunoassays for determining binding are well known to those of skill in the art.
  • a preferred immunoassay is a competitive immunoassay as discussed, infra.
  • the proteins of the present invention can be employed as immunogens for constructing antibodies immunoreactive to a protein of the present invention for such exemplary utilities as immunoassays or protein purification techniques.
  • an hCRSPl polypeptide of the present invention can include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation, as specified herein.
  • Functional analogs of hCRSPl polypeptide (s) are typically generated by deletion, insertion, or substitution of a single (or few) amino acid residues.
  • Functional analogs of the polypeptide of SEQ ID NO : 4 or 5 may be those in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code. Substitution modifications can generally be made in accordance with the following Table. Table 1
  • the number of amino acid substitutions a skilled artisan would make depends on many factors, including those described above. Generally speaking, the number of amino acid substitutions, insertions or deletions for any given hCRSPl polypeptide will not be more than 40, 30, 20, 10, 5, or 3, such as 1-30 or any range or value therein, as specified herein.
  • Amino acids in an hCRSPl polypeptide of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule . The resulting mutant molecules are then tested for biological activity. Sites that are critical for ligand-protein binding can also be identified by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith, et al . , J. Mol . Biol. 224:899-904 (1992) and de Vos, et al . , Science 255:306-312 (1992) ) .
  • Non-limiting mutants that can enhance or maintain at least one of the listed activities include any of the polypeptides described herein further comprising at least one mutation corresponding to at least one substitution, insertion or deletion selected from the group consisting of 66Y, 75A, 89K, 118S, 199F, 120T, 126T, 137D, 139K, 144S, 145S, 147Q, 148N, 152V, 154S, 156P, 158T, 1590, 161P, 162L, 163S, 164Q, 165G, 166N, 167S, 169R, 170K, 228R, 230N, 231P, 232R, 233E, 234G, 2351, 242P, 253Q, 269L, 277V, 278M, 280S, 282E, 283S, 284D, 302P, 306E, 307G, 310M, 3131, 316L, 321T, 351P, 3
  • Covalent modifications of hCRSPl polypeptides are also included within the scope of this invention.
  • One type of covalent modification includes reacting targeted amino acid residues of an hCRSPl polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of an hCRSPl polypeptide.
  • Derivatization with bifunctional agents is useful, for instance, for crosslinking hCRSPl to a water-insoluble support matrix or surface for use in the method for purifying anti- hCRSPl antibodies, and vice-versa.
  • crosslinking agents include, e.g., 1 .1 -bis (diazo-acetyl) -2-phenylethane, glutaraldehyde, N-hydroxy-succinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoester ⁇ .
  • disuccinimidyl esters such as 3,3'- dithiobis- (succinimidyhCRSPlroprionate) .
  • bifunctional maleimides such as bis-N-maleimidol , 8-octane and agents such as methyl-3- [ (p-azidophenyl) -dithiohCRSPlroprioimidate.
  • Another type of covalent modification of the hCRSPl polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide.
  • "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence hCRSPl polypeptide. and/or adding one or more glycosylation sites that are not present in the native sequence hCRSPl polypeptide.
  • the phrase includes qualitative changes in the glycosylation of the native proteins as well as changes in the nature and proportions of the various carbohydrate moieties present.
  • hCRSPl variant that would be useful in the methods of the present invention is a hCRSPl polypeptide as defined herein further comprising one or more amino acid substitutions that result in an altered glycosylation pattern as compared to the corresponding unsubstituted hCRSPl polypeptide.
  • the term ".W-glycosyled polypeptide” refers to polypeptides having one or more NXS/T motifs in which the nitrogen atom in the side chain amide of the asparagine is covalently bonded to a glycosyl group.
  • "X" refers to any naturally occurring amino acid residue except proline.
  • the "naturally occurring amino acids” are glycine, alanine, valine, leucine, isoleucine, proline, serine, threonine, cysteine, methionine, lysine, arganine, glutamic acid, asparatic acid, glutamine, asparagine, phenylalanine, histidine, tyrosine and tryptophan.
  • i ⁇ 7-glycosylated proteins are optionally O-glycosylation.
  • O-glycosylated polypeptide refers to polypeptides having one or more serines and/or threonine in which the oxygen atom in the side chain is covalently bonded to a glycosyl group.
  • O-Glycosylated proteins are optionally N-glycosylation .
  • Glycosylated polypeptides can be prepared recombinantly by expressing a gene encoding a polypeptide in a suitable mammalian host cell, resulting in glycosylation of side chain amides found in accessible NXT/S motifs on the polypeptide surface and/or of side chain alcohols of surface accessible serines and threonines .
  • Unglycosylated polypeptides can be prepared recombinantly by expressing a gene encoding a polypeptide in a suitable prokaryotic host cell.
  • the hCRSPl polypeptides and hCRSPl fragments and/or hCRSPl variants of the present invention can also be glycosylated or unglycosylated.
  • a glycosylated polypeptide is modified with one or more monosaccharides or oligosaccharides .
  • a monosaccharide is a chiral polyhydroxyalkanol or polyhydroxyalkanone which typically exists in hemiacetal form.
  • An "oligosaccharide” is a polymer of from about 2 to about 18 monosaccharides which are generally linked by acetal bonds .
  • One type of glycosyl group commonly found in glycosylated proteins is N-acetylneuraminic acid.
  • a glycosylated polypeptide can be -glycosylated and/or O- glycosylated, preferably N-glycosylated .
  • Addition of glycosylation sites to hCRSPl polypeptides may be accomplished by altering the amino acid sequence thereof.
  • the alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence hCRSPl polypeptide (for O-linked glycosylation sites).
  • the hCRSPl amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the hCRSPl polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
  • Another means of increasing the number of carbohydrate moieties on the hCRSPl polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87105330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).
  • Removal of carbohydrate moieties present on the hCRSPl polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation.
  • Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al . , Arch. Biochem. Biophys . , 259:52 (1987) and by Edge et al . , Anal. Biochem. ) m:131 (1981) .
  • Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of. endo- and exo-glycosidases as described by Thotakura et al . , Meth. Enzymof., 138:350 (1987).
  • Another type of covalent modification of hCRSPl comprises linking the hCRSPl polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4640,835; 4,496,689: 4,301,144; 4.670,417; 4.791,192 or 4,179,337, Mumtaz and Bachhawat, Indian Journal of Biochemistry and Biophysics 28:346 (1991) and Franciset al . , International Journal of Hematology 68 : 1 (1998), the entire teachings of which are incorporated herein by reference.
  • nonproteinaceous polymers e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes
  • the hCRSPl variants useful in the methods of the present invention also include hCRSPl polypeptides as defined herein further comprising one or more polyethylene glycol groups (hereinafter "PEG" groups) .
  • PEG groups generally have a molecular weight between about 5000 and 40,000 atomic mass units.
  • hCRSPl polypeptides of the present invention may also be modified in a way to form chimeric molecules comprising an hCRSPl polypeptide fused to another heterologous polypeptide or amino acid sequence.
  • hCRSPl fusion proteins represent a hybrid protein molecule not found in nature comprising a translational fusion or enzymatic fusion in which two or more different proteins, fragments, or variants thereof are covalently linked on a single polypeptide chain.
  • a chimeric molecule comprises a fusion of an hCRSPl polypeptide with a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind.
  • the epitope tag is generally placed at the amino- or carboxyl- terminus of the hCRSPl polypeptide. The presence of such epitope-tagged forms of an hCRSPl polypeptide can be detected using an antibody against the tag polypeptide.
  • hCRSPl polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.
  • Human serum albumin, the C-terminal domain of thrombopoietin, the C-terminal extension peptide of hCG, and/or a Fc fragment are examples of proteins which could be fused with hCRSPl polypeptides, hCRSPl fragments and/or hCRSPl variants for use in the present invention.
  • Fc fragment of an antibody has the meaning commonly given to the term in the field of immunology.
  • this term refers to an antibody fragment which binds complement and is obtained by removing the two antigen binding regions (the Fab Fragments) from the antibody.
  • the Fc fragment is formed from approximately equal sized fragments from both heavy chains, which associate through non-covalent interactions and disulfide bonds.
  • the Fc Fragment includes the hinge regions and extends through the C H 2 and C H 3 domains to the C-terminus of the antibody.
  • fusion proteins can be secreted by virtue of heterologous secretion signals in regions that can be removed prior to final preparation of the polypeptide.
  • Such methodologies are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18, the entire relevant teachings of which are incorporated herein by reference.
  • the hCRSPl gene can be modified at the 5 ' end to incorporate several histidine residues at the amino terminus of the hCRSPl protein resulting from its expression.
  • This "histidine tag” enables a single-step protein purification method referred to as “immobilized metal ion affinity chromatography” (IMAC) , essentially as described in U.S. Patent 4,569,794, which hereby is incorporated by reference.
  • IMAC immobilized metal ion affinity chromatography
  • Antigenic/Epitope Comprising hCRSPl Peptide and Polypeptides
  • the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention according to methods well known in the art (See, e.g., Colligan, et al,. ed. , Current
  • the epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide described herein.
  • An "immunogenic epitope" can be defined as a part of a polypeptide that elicits an antibody response when the whole polypeptide is the immunogen.
  • a region of a polypeptide molecule to which an antibody can bind is defined as an "antigenic epitope.”
  • the number of immunogenic epitopes of a polypeptide generally is less than the number of antigenic epitopes. See, for instance, Geysen, et al . , Proc . Natl. Acad. Sci. USA 81:3998-4002 (1983) .
  • peptides or polypeptides bearing an antigenic epitope i.e., that contain at least a portion of a region of a polypeptide molecule to which an antibody can bind
  • relatively short synthetic peptides that mimic part of a polypeptide sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked polypeptide. See, for instance, J. G. Sutcliffe, et al . , "Antibodies that react with preidentified sites on polypeptides," Science 219:660-666 (1983) .
  • Antigenic epitope-bearing peptides and polypeptides of the invention are useful to raise antibodies, including monoclonal antibodies, or screen antibodies, including fragments or single chain antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson, et al . , Cell 37:767-778 (1984) at 777.
  • Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least five, more preferably at least nine, and most preferably between at least about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.
  • the epitope-bearing peptides and polypeptides of the invention can be produced by any conventional means.
  • R. A. Houghten "General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids," Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985) .
  • This “Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U.S. Patent No. 4,631,211 to Houghten, et al . (1986).
  • hCRSPl polypeptides of the present invention and the epitope- bearing fragments thereof described above can be combined with parts of the constant domain of immunoglobulins (IgG) , resulting in chimeric polypeptides.
  • IgG immunoglobulins
  • These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4- polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker, et al . , Nature 331:84-86 (1988)).
  • Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric hCRSPl polypeptide or polypeptide fragment alone (Fountoulakis, et al . , J. Biochem. 270:3958-3964 (1995)).
  • the polypeptides of this invention and fragments thereof may be used in the production of antibodies.
  • antibody as used herein describes antibodies, fragments of antibodies (such as, but not limited, to Fab, Fab 1 , Fab2 ' , and Fv fragments), and modified versions thereof, as well known in the art (e.g., chimeric, humanized, recombinant, veneered, resurfaced or CDR- grafted) .
  • antibody is meant to include polyclonal antibodies, monoclonal antibodies (MAbs), chimeric antibodies, single-chain polypeptide binding molecules, and anti-idiotypic (anti-id) antibodies.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immu- nised with an antigen while monoclonal antibodies (MAbs) are a substantially homogeneous population of antibodies to specific antigens.
  • MAbs monoclonal antibodies
  • Polyclonal and MAbs may be obtained by methods known to those skilled in the art (for MAbs, see, for example, Kohler et al . , Nature 256 : 495-497 (1975), Colligan, supra., and U.S. Pat. No. 4,376,110).
  • Single chain antibodies and libraries thereof are yet another variety of genetically engineered antibody technology that is well known in the art.
  • Single chain antibody technology involves covalently joining the binding regions of heavy and light chains to generate a single polypeptide chain. The binding specificity of the intact antibody molecule is thereby reproduced on a single polypeptide chain.
  • MAbs may be of any immuno-globulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • the hybridoma producing the MAbs of this invention may be cultivated in vi tro or in vivo . Production of high titers of MAbs in vivo makes this the presently preferred method of production. Briefly, cells from the individual hybridomas are injected intraperitoneally into pristane-primed BALB/C mice to produce ascites fluid containing high concentrations of the desired MAbs.
  • MAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.
  • Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having variable region derived from a murine MAb and a human immunoglobulin constant region. Chimeric antibodies and methods for their production are known in the art (Cabilly et al . , Proc . Natl . Acad. Sci . (USA) 71-3273-3277 (1984); Morrison et al . , Proc . Natl . Acad . Sci . (USA) 81:6851-6855 (1984); Boulianne et al . , Nature 312:643646 (1984); Cabilly et al . , European Patent Application 125023 (published Nov. 14, 1984); Neuberger et al .
  • the most preferred method of generating MAbs to the polypeptides and glycopeptides of the present invention comprises producing said MAbs in a transgenic mammal modified in such a way that they are capable of producing fully humanized MAbs upon antigenic challenge.
  • Fully humanized MAbs and methods for their production are generally known in the art (PCT/WO9634096 , PCT/W09633735 , and PCT/W09824893 ) . These documents are hereby incorporated by reference.
  • An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody.
  • An anti-Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g. mouse strain) as the source of the MAb with the MAb to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody) .
  • the anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody.
  • the anti-anti-Id may be epitopically identical to the original MAb which induced the anti-Id.
  • MAbs generated against a hCRSP protein or glycoprotein of the present invention may be used to induce anti-Id antibodies in suitable animals, such as BALB/C mice and/or any transgenically altered mouse capable of producing fully humanized MAbs. Spleen cells from such immunized mice are used produce anti-Id hybridomas secreting anti-Id MAbs.
  • the anti-Id MAbs can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize additional similar mice. Sera from these mice will contain anti-anti-Id antibodies that have the binding properties of the original MAb specific for a hCRSP epitope.
  • KLH keyhole limpet hemocyanin
  • Sera from these mice will contain anti-anti-Id antibodies that have the binding properties of the original MAb specific for a hCRSP epitope.
  • the anti-Id MAbs thus have their own idiotypic epitopes, or "idiotopes" structurally similar to the epitope being evaluated, such as a hCRSP protein or glycoprotein.
  • antibody is also meant to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab'),, which are capable of binding antigen.
  • Fab and F(ab') fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al . , J. Nucl. Med. 24:316-325 (1983)).
  • Fab and F(ab')a and other fragments of the antibodies useful in the present invention may be used for the detection and quantitation of a hCRSP protein or glycoprotein according to methods disclosed herein for intact antibody molecules .
  • Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments).
  • a polypeptide used as an immunogen may be modified or administered in an adjuvant, by subcutaneous or intraperitoneal injection into, for example, a mouse or a rabbit.
  • spleen cells from immunized animals are removed, fused with myeloma or other suitable known cells, and allowed to become monoclonal antibody producing hybridoma cells in the manner known to the skilled artisan.
  • Hybridomas that secrete a desired antibody molecule can be screened by a variety of well known methods, for example ELISA assay, Western blot analysis, or radioimmunoassay (Lutz, et al . Exp . Cell Res. 175:109-124 (1988); Monoclonal Antibodies: Principles & Applications, Ed. J. R. Birch & E. S. Lennox, Wiley-Liss (1995); Colligan, supra).
  • Antibodies included in this invention are useful in diagnostics, therapeutics or in diagnostic/ therapeutic combinations.
  • polypeptides of this invention or suitable fragments thereof can be used to generate polyclonal or monoclonal antibodies, and various inter- species hybrids, or humanized antibodies, or antibody fragments, or single-chain antibodies.
  • the present invention relates to a method for detecting the presence of or measuring the quantity of a hCRSP protein or glycoprotein in a cell, comprising:
  • labeled antibodies are desirable.
  • Procedures for labeling antibody molecules are widely known, including for example, the use of radioisotopes , affinity labels, such as biotin or avidin, enzymatic labels, for example horseradish peroxidase, and fluorescent labels, such as FITC or rhodamine (see, e.g., Colligan, supra). Labeled antibodies are useful for a variety of diagnostic applications.
  • the present invention relates to the use of labeled antibodies to detect the presence of an hCRSPl polypeptide.
  • the antibodies could be used in a screen to identify potential modulators of an hCRSPl polypeptide.
  • the antibody or compound to be tested is labeled by any suitable method.
  • Competitive displacement of an antibody from an antibody-antigen complex by a test compound such that a test compound-antigen complex is formed provides a method for identifying compounds that bind hCRSPl.
  • the recombinant gene construct may be composed of regulatory DNA sequences that belong to the native hCRSPl gene or those which are derived from an alternative source. These regulatory sequences are functionally linked to the hCRSPl coding region, resulting in the constitutive and/or regulatable expression of hCRSPl in the body of the transgenic non-human mammal.
  • the most important of such regulatory sequences is the promoter. Promoters are defined in this context as any and all DNA elements necessary for the functional expression of a gene. Promoters drive the expression of structural genes and may be modulated by inducers and repressors.
  • the genome of the transgenic mammal contains at least 30 copies of a transgene . More preferably, the genome of the transgenic mammal contains at least 50 copies, and may contain 100-200 or more copies of the transgene.
  • said nucleic acid is introduced into said mammal at an embryonic stage, preferably the 1-1000 cell or oocyte stage, and, most preferably not later than about the 64-cell stage. Most preferably the transgenic mammal is homozygous for the transgene .
  • chimeric non-human mammals in which fewer than all of the somatic and germ cells contain a DNA construct comprising a nucleic acid encoding a hCRSPl polypeptide of the present invention.
  • Contemplated chimeric non-human mammals include animals produced when fewer than all of the cells of the morula are transfected in the process of producing the transgenic animal .
  • Transgenic and chimeric non-human mammals having human cells or tissue engrafted therein are also encompassed by the present invention.
  • Methods for providing chimeric non-human mammals are provided, e.g., in U.S. Serial Nos. 07/508,225, 07/518,748, 07/529,217, 07/562,746, 07/596,518, 07/574,748, 07/575,962, 07/207,273, 07/241,590 and 07/137,173, which are entirely incorporated herein by reference, for their description of how to engraft human cells or tissue into non- human mammals.
  • genetic constructs comprising at least one of the hCRSPl nucleic acid sequences as defined herein may be used to create transgenic "knockouts" of the hCRSPl gene.
  • the present invention also provides a transgenic animal which has been engineered by homologous recombination to be deficient in the expression of the endogenous CRSPl gene.
  • the invention provides a method of producing an heterozygous or homozygous transgenic animal deficient in or lacking functional CRSPl proteins, respectfully, said method comprising: a) obtaining a DNA construct comprising a disrupted hCRSPl gene, wherein said disruption is by the insertion of an heterologous marker sequence; b) introducing said DNA construct into an ES cell of said animal such that the endogenous CRSPl gene is disrupted by homologous recombination; c) selecting ES cells comprising said disrupted allele; d) incorporating the ES cells of step c) into a mouse embryo ; e) transferring said embryo into a pseudopregnant animal of the said species; f) developing said embryo into a viable offspring; g) screening offspring to identify heterozygous animal comprising said disrupted CRSPl gene; and h) if desired, breeding said heterozygous animal to produce homozygous transgenic animals of said species, wherein the said homozygous
  • Transgenic and chimeric non-human mammals of the present invention may be used for analyzing the consequences of over- expression of at least one hCRSPl polypeptide in vivo. Such animals are also useful for testing the effectiveness of therapeutic and/or diagnostic agents, either associated or unassociated with delivery vectors or vehicles, which preferentially bind to an hCRSPl polypeptide of the present invention or act to indirectly modulate hCRSPl activity.
  • hCRSPl transgenic non-human mammals are useful as an animal models in both basic research and drug development endeavors.
  • Transgenic animals carrying at least one hCRSPl polypeptide or nucleic acid can be used to test compounds or other treatment modalities which may prevent, suppress, or cure a pathology or disease associated with at least one of the above mentioned hCRSPl activities. Such transgenic animals can also serve as a model for the testing of diagnostic methods for those same diseases. Furthermore, tissues derived from hCRSPl transgenic non-human mammals are useful as a source of cells for cell culture in efforts to develop in vi tro bioassays to identify compounds that modulate hCRSPl activity or hCRSPl dependent signaling. Accordingly, another aspect of the present invention contemplates a method of identifying compounds efficacious in the treatment of at least one previously described disease or pathology associated with CRSP activity.
  • a non- limiting example of such a method comprises: a) generating an hCRSPl transgenic non-human animal which is, as compared to a wild-type animal, pathologically distinct in some detectable or measurable manner from wild- type version of said non-human mammal; b) exposing said transgenic animal to a compound, and; c) determining the progression of the pathology in the treated transgenic animal, wherein an arrest, delay, or reversal in disease progression in transgenic animal treated with said compound as compared to the progression of the pathology in an untreated control animals is indicative that the compound is useful for the treatment of said pathology
  • Another embodiment of the present invention provides a method of identifying compounds capable of inhibiting hCRSPl activity in vivo and/or in vi tro wherein said method comprises : a) administering an experimental compound to an hCRSPl transgenic non-human animal, or tissues derived therefrom, exhibiting one or more physiological or pathological conditions attributable to the overexpression of an hCRSPl trans
  • Another embodiment of the invention provides a method for identifying compounds capable of overcoming deficiencies in hCRSPl activity in vivo or in vi tro wherein said method comprises : a) administering an experimental compound to an hCRSPl transgenic non-human animal, or tissues derived therefrom, exhibiting one or more physiological or pathological conditions attributable to the disruption of the endogenous CRSPl gene; and b) observing or assaying said animal and/or animal tissues to detect changes in said physiological or pathological condition or conditions.
  • Various means for determining a compound's ability to modulate hCRSPl in the body of the transgenic animal are consistent with the invention. Observing the reversal of a pathological condition in the transgenic animal after administering a compound is one such means. Another more preferred means is to assay for markers of hCRSPl activity in the blood of a transgenic animal before and after administering an experimental compound to the animal. The level of skill of an artisan in the relevant arts readily provides the practitioner with numerous methods for assaying physiological changes related to therapeutic modulation of hCRSPl activity.
  • the invention further provides for the use of a hCRSPl agonist, hCRSPl antagonist, hCRSPl polypeptide, hCRSPl nucleic acid, or hCRSPl antibody in the manufacture of a medicament for the treatment or prevention of tissue damage, cell proliferation or tissue growth disorders such as nerve damage, neurodegeneration, trigeminal neuralgia, glossopharyngeal neuralgia, Bell's Palsy, myasthenia gravis, muscular dystrophy, progressive muscular atrophy, progressive bulbar inherited muscular atrophy, herniated, ruptured or prolapsed invertebrae disk, cervical spondylosis, plexus disorders, thoracic outlet destruction, peripheral neuropathy, such as those caused by lead, dapsone, ticks or porphyria, peripheral myelin disorders, Alzheimer's disease, Gullain-Barre syndrome, Parkinson's disease, Parkinsonian disorders, ALS, multiple sclerosis, other central myelin disorders, stroke, ischemia associated with stroke
  • the present invention contemplates the use of hCRSPl agonists, hCRSPl antagonists, hCRSPl polynucleotides, hCRSPl polypeptides, hCRSPl variants, and hCRSPl antibodies to treat metabolic diseases such as obesity, cachexia, bulimia, anorexia, and/or disorders commonly associated with these conditions such as dyslipidemia and diabetes, especially non-insulin dependent diabetes.
  • metabolic diseases such as obesity, cachexia, bulimia, anorexia, and/or disorders commonly associated with these conditions such as dyslipidemia and diabetes, especially non-insulin dependent diabetes.
  • a subject e.g, a mammalian subject, such as a human, a domesticated animal, or an animal used in agriculture.
  • the present invention includes the administration of hCRSPl agonists, hCRSPl antagonists, hCRSPl polynucleotides, hCRSPl polypeptides, hCRSPl variants, and/or hCRSPl antibodies for veterinary or human therapeutic uses
  • the invention provides methods to induce or inhibit angiogenesis, neovascular- ization, tumorigenesis, cataractogenesis, wound healing, growth-factor mediated chemotaxis, neurite outgrowth and/or neurite adhesion comprising administering to patient a pharmaceutically acceptable composition comprising a hCRSPl nucleic acid, polypeptide, cells, and/or antibody described herein and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable composition comprising a hCRSPl nucleic acid, polypeptide, cells, and/or antibody described herein and a pharmaceutically acceptable carrier.
  • an effective amount of hCRSPl polypeptide, hCRSPl variant, and/or hCRSPl antibody is administered to a mammal in need thereof in a dose between about 0.1 and 1000 ⁇ g/kg body weight.
  • the hCRSPl polypeptides, hCRSPl variants, and/or hCRSPl antibodies as defined herein can be administered in multiple doses per day, in single daily doses, in weekly doses, or at any other regular interval.
  • the amount per administration and frequency of administration will be determined by a physician and depend on such factors as the nature and severity of the disease, and the age and general health of the patient.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising as the active agent an hCRSPl agonist, hCRSPl antagonist, hCRSPl polynucleotide, hCRSPl polypeptide, hCRSPl fragment, hCRSPl variant, and/or hCRSPl antibody, and/or a pharmaceutically acceptable non-toxic salt thereof, and a pharmaceutically acceptable solid or liquid carrier.
  • compounds comprising at least one hCRSPl agonist, hCRSPl antagonist, hCRSPl polynucleotide, hCRSPl polypeptide, hCRSPl fragment, hCRSPl variant, and/or hCRSPl antibody can be admixed with conventional pharmaceutical carriers and excipients, and used in the form of tablets, capsules, elixirs, suspensions, syrups, wafers, parenteral formulations, and the like.
  • compositions comprising at least one hCRSPl agonist, hCRSPl antagonist, hCRSPl polynucleotide, hCRSPl polypeptide, hCRSPl fragment, hCRSPl variant, and/or hCRSPl antibody, will contain from about 0.1% to 90% by weight of the active compound, and more generally from about 10% to 30%.
  • the compositions may contain common carriers and excipients such as corn-starch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, and alginic acid.
  • the total pharmaceutically effective amount of a hCRSPl agonist, hCRSPl antagonist, hCRSPl polynucleotide, hCRSPl polypeptide, hCRSPl fragment, hCRSPl variant, and/or hCRSPl antibody administered parenterally per dose will be in the range of about 1 ⁇ g/kg/day to 10 mg/kg/day of patient body weight, particularly 2 mg/kg/day to 8 mg/kg/day, more particularly 2 mg/kg/day to 4 mg/kg/day, even more particularly 2.2 mg/kg/day to 3.3 mg/kg/day, and finally 2.5 mg/kg/day, although, as noted above, this will be subject to therapeutic discretion.
  • this dose is at least 0.01 mg/kg/day.
  • a hCRSPl agonist, hCRSPl antagonist, hCRSPl polynucleotide, hCRSPl polypeptide, hCRSPl fragment, hCRSPl variant, and/or hCRSPl antibody is typically administered at a dose rate of about 1 ⁇ g/kg/hour to about 50 ⁇ g/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.
  • compositions containing a hCRSPl agonist, hCRSPl antagonist, hCRSPl polynucleotide, hCRSPl polypeptide, hCRSPl fragment, hCRSPl variant, and/or hCRSPl antibody may be administered orally, rectally, intracranially, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), transdermally, intrathecally, bucally, or as an oral or nasal spray.
  • pharmaceutically acceptable carrier is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • parenteral includes, but is not limited to, modes of administration which include intravenous, intramuscular, intraperitoneal , intrasternal, subcutaneous and intra-articular injection, infusion and implants comprising a hCRSPl agonist, hCRSPl antagonist, hCRSPl polynucleotide, hCRSPl polypeptide, hCRSPl fragment, hCRSPl variant, and/or hCRSPl antibody.
  • the compounds can be formulated for oral or parenteral administration.
  • a preferred parenteral formulation for subcutaneous administration would comprise a buffer (phosphate, citrate, acetate, borate, TRIS) , salt (NaCl, KC1) , divalent metal (Zn, Ca) , and isotonicty agent (glycerol, mannitol), detergent (Polyoxyethylene sorbitan fatyy acid esters, poloxamer, ddicusate sodium, sodium lauryl sulfate) , antioxidants (ascorbic acid) , and antimicrobial agent (phenol, m-cresol, alcohol, benzyl alcohol, butyhCRSPlarben, methyhCRSPlaraben, ethyhCRSPlaraben, chlorocresol , phenoxyethanol,phenylethyl alcohol, propyhCRSPlaraben.
  • a buffer phosphate, citrate, acetate, borate, TRIS
  • salt NaCl, KC
  • a hCRSPl agonist, hCRSPl antagonist, hCRSPl polynucleotide, hCRSPl polypeptide, hCRSPl fragment, hCRSPl variant, and/or hCRSPl antibody is administered in commonly used intravenous fluid (s) and administered by infusion.
  • s intravenous fluid
  • Such fluids for example, physiological saline, Ringer's solution or 5% dextrose solution can be used.
  • a sterile formulation preferably a suitable soluble salt form of a hCRSPl agonist, hCRSPl antagonist, hCRSPl polynucleotide, hCRSPl polypeptide, hCRSPl fragment, hCRSPl variant, and/or hCRSPl antibody such as the hydrochloride salt
  • a pharmaceutical diluent such as pyrogen-free water (distilled) , physiological saline or 5% glucose solution.
  • a suitable insoluble form of the compound may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e.g. an ester of a long chain fatty acid such as ethyl oleate.
  • a hCRSPl agonist, hCRSPl antagonist, hCRSPl polynucleotide, hCRSPl polypeptide, hCRSPl fragment, hCRSPl variant, and/or hCRSPl antibody is also suitably administered by sustained-release systems.
  • sustained-release compositions include semi- permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules .
  • Sustained-release matrices include polylactides (U.S. Pat. No. 3,773.919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. et al .
  • sustained-release compositions also include liposomally entrapped modified hCRSPl polypeptides and/or fragments thereof and/or variants thereof.
  • Such liposomes are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al . , Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EDP 143,949; EP 142,641; Japanese Pat. Appl . 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.
  • the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol . percent cholesterol, the selected proportion being adjusted for the optimal therapy.
  • the hCRSPl agonist, hCRSPl antagonist, hCRSPl polynucleotide, hCRSPl polypeptide, hCRSPl fragment, hCRSPl variant, and/or hCRSPl antibody is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion) , with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to polypeptides .
  • the formulations are prepared by contacting hCRSPl agonist, hCRSPl antagonist, hCRSPl polynucleotide, hCRSPl polypeptide, hCRSPl fragment, hCRSPl variant, and/or hCRSPl antibody uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation.
  • the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes .
  • the carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability.
  • additives such as substances that enhance isotonicity and chemical stability.
  • Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinyhCRSPlyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharide ⁇ , and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol
  • a hCRSPl agonist, hCRSPl antagonist, hCRSPl polynucleotide, hCRSPl polypeptide, hCRSPl fragment, hCRSPl variant, and/or hCRSPl antibody is typically formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of salts of the particular active ingredient (s) . Compositions to be used for therapeutic administration must be sterile.
  • Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes) .
  • Therapeutic compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • compositions comprising a hCRSPl agonist, hCRSPl antagonist, hCRSPl polynucleotide, hCRSPl polypeptide, hCRSPl fragment, hCRSPl variant, and/or hCRSPl antibody ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution.
  • a lyophilized formulation 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous solution of one of the hCRSPl polypeptides and/or fragments and/or hCRSPl variants of the present invention, and the resulting mixture is lyophilized.
  • the infusion solution is prepared by reconstituting the lyophilized polypeptide using bacteriostatic Water-for-Injection.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Associated with such container ( s ) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • a hCRSPl agonist, hCRSPl antagonist, hCRSPl polynucleotide, hCRSPl polypeptide, hCRSPl fragment, hCRSPl variant, and/or hCRSPl antibody may be administered in the methods of the present invention in combination with other therapeutic compounds.
  • Administration in combination with one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
  • Nucleic acids encoding the hCRSPlXX polypeptides may also be used in gene therapy.
  • genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example, for replacement of a defective gene.
  • Gene therapy includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA.
  • a ntisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo.
  • oligonucleotides can be imported into cells where act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane (Za ecnik et al . , Proc. Natl. Acad Sci. USA 83: 4143-4146 [1986] ) .
  • the oligonucleotides can be modified to enhance their uptake, e.g., by substituting their negatively charged phosphodiester groups by uncharged groups.
  • There are a variety of techniques available for introducing nucleic acids into viable cells The techniques vary depending upon whether the nucleic acid is transferred into cultured cell in vitro, or in vivo in the cells of the intended host.
  • nucleic acid into mammalian cells in vitro
  • liposomes include liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc.
  • the currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau, et al . , Trends in Biotechnology 11: 205-
  • nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cells, etc.
  • an agent that targets the target cells such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cells, etc.
  • proteins which bind to a cell surface membrane protein associated with endocytosis may by used for targeting and/or to facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, protein that target intracellular localization and enhance intracellular half-life.
  • the technique of receptor-mediated endocytosis is described, for example by Wu et al . , J. Biol. Chem. 262: 4429-4432 (1987).
  • Anderson et al . , Science 256: 808- 813 (1992) see Anderson et al . , Science 256:
  • Example 1 Expression and Purification of an hCRSPl Polypeptide in E. coli
  • the bacterial expression vector pQE60 is used for bacterial expression in this example. (QIAGEN, Inc., Chatsworth, CA) .
  • pQE60 encodes ampicillin antibiotic resistance ("Ampr") and contains a bacterial origin of replication ("ori"), an IPTG inducible promoter, a ribosome binding site (“RBS”), six codons encoding histidine residues that allow affinity purification using nickel-nitrilo-tri- acetic acid ( "Ni-NTA” ) affinity resin sold by QIAGEN, Inc., and suitable single restriction enzyme cleavage sites.
  • a DNA fragment encoding a polypeptide can be inserted in such a way as to produce that polypeptide with the six His residues (i.e., a "6 X His tag") covalently linked to the carboxyl terminus of that polypeptide.
  • a polypeptide coding sequence can optionally be inserted such that translation of the six His codons is prevented and, therefore, a polypeptide is produced with no 6 X His tag.
  • the nucleic acid sequence encoding the desired portion of an hCRSPl polypeptide lacking the hydrophobic leader sequence is amplified from the deposited cDNA clone using PCR oligonucleotide primers (based on the sequences presented, e.g., as presented in at least one of SEQ ID NOS : 1 , 2, 3) , which anneal to the amino terminal encoding DNA sequences of the desired portion of an hCRSPl polypeptide and to sequences in the deposited construct 3 ' to the cDNA coding sequence. Additional nucleotides containing restriction sites to facilitate cloning in the pQE60 vector are added to the 5' and 3' sequences, respectively.
  • the 5' and 3' primers have nucleotides corresponding or complementary to a portion of the coding sequence of an hCRSPl, e.g., as presented in at least one of SEQ ID NOS : 1 , 2, 3, according to known method steps.
  • SEQ ID NOS : 1 , 2, 3, SEQ ID NOS : 1 , 2, 3, according to known method steps.
  • the point in a polypeptide coding sequence where the 5 ' primer begins can be varied to amplify a desired portion of the complete polypeptide shorter or longer than the mature form.
  • the amplified hCRSPl nucleic acid fragments and the vector pQE60 are digested with appropriate restriction enzymes and the digested DNAs are then ligated together.
  • Insertion of the hCRSPl DNA into the restricted pQE60 vector places an hCRSPl polypeptide coding region including its associated stop codon downstream from the IPTG-inducible promoter and in-frame with an initiating AUG codon.
  • the associated stop codon prevents translation of the six histidine codons downstream of the insertion point.
  • E. coli strain Ml5/rep4 containing multiple copies of the plasmid pREP4 , which expresses the lac repressor and confers kanamycin resistance ("Kanr"), is used in carrying out the illustrative example described herein.
  • This strain which is only one of many that are suitable for expressing hCRSPl polypeptide, is available commercially from QIAGEN, Inc. Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis, PCR and DNA sequencing.
  • Clones containing the desired constructs are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 ⁇ g/ml) and kanamycin (25 ⁇ g/ml) .
  • the O/N culture is used to inoculate a large culture, at a dilution of approximately 1:25 to 1:250.
  • the cells are grown to an optical density at 600 nm ("OD600") of between 0.4 and 0.6.
  • Isopropyl-b-D-thiogalactopyranoside (“IPTG”) is then added to a final concentration of 1 mM to induce transcription from the lac repressor sensitive promoter, by inactivating the lad repressor.
  • Cells subsequently are incubated further for 3 to 4 hours. Cells then are harvested by centrifugation.
  • the cells are then stirred for 3-4 hours at 4°C in 6M guanidine-HCl, pH8.
  • the cell debris is removed by centrifugation, and the supernatant containing the hCRSPl is dialyzed against 50 mM Na-acetate buffer pH6, supplemented with 200 mM NaCl .
  • a polypeptide can be successfully refolded by dialyzing it against 500 mM NaCl, 20% glycerol, 25 mM Tris/HCl pH7.4, containing protease inhibitors .
  • the protein is made soluble according to known method steps. After renaturation the polypeptide is purified by ion exchange, hydrophobic interaction and size exclusion chromatography. Alternatively, an affinity chromatography step such as an antibody column is used to obtain pure hCRSPl polypeptide. The purified polypeptide is stored at 4°C or frozen at -40°C to -120°C.
  • Example 2 Cloning and Expression of an hCRSPl Polypeptide in a Baculovirus Expression System
  • the plasmid shuttle vector pA2 GP is used to insert the cloned DNA encoding the mature polypeptide into a baculovirus to express an hCRSPl polypeptide, using a baculovirus leader and standard methods as described in Summers, et al . , A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987) .
  • This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the secretory signal peptide (leader) of the baculovirus gp67 polypeptide and convenient restriction sites such as BamHI, Xba I and Asp718.
  • the polyadenylation site of the simian virus 40 ("SV40") is used for efficient polyadenylation.
  • the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene.
  • the inserted genes are flanked on both sides by viral sequences for cell- mediated homologous recombination with wild-type viral DNA to generate viable virus that expresses the cloned polynucleotide .
  • baculovirus vectors are used in place of the vector above, such as pAc373, pVL941 and pAcIMl, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required.
  • Such vectors are described, for instance, in Luckow, et al . , Virology 170:31-39.
  • the cDNA sequence encoding the mature hCRSPl polypeptide in the deposited or other clone, lacking the AUG initiation codon and the naturally associated nucleotide binding site, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene.
  • Non- limiting examples include 5' and 3' primers having nucleotides corresponding or complementary to a portion of the coding sequence of an hCRSPl polypeptide, e.g., as presented in at least one of SEQ ID NOS:l, 2, 3, according to known method steps.
  • the amplified fragment is isolated from a 1% agarose gel using a commercially available kit (e.g., "Geneclean, " BIO 101 Inc., La Jolla, CA) .
  • the fragment then is then digested with the appropriate restriction enzyme and again is purified on a 1% agarose gel.
  • This fragment is designated herein "Fl”.
  • the plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art.
  • the DNA is then isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, CA) . This vector DNA is designated herein "VI” .
  • E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, CA) cells are transformed with the ligation mixture and spread on culture plates.
  • Bacteria are identified that contain the plasmid with the hCRSPl gene using the PCR method, in which one of the primers that is used to amplify the gene and the second primer is from well within the vector so that only those bacterial colonies containing the hCRSPl gene fragment will show amplification of the DNA.
  • the sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is designated herein pBac hCRSPl .
  • plasmid pBachCRSPl Five ⁇ g of the plasmid pBachCRSPl is co-transfected with 1.0 ⁇ g of a commercially available linearized baculovirus DNA ( "BaculoGoldTM baculovirus DNA", Pharmingen, San Diego, CA) , using the lipofection method described by Feigner, et al . , Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987) .
  • baculovirus DNA "BaculoGoldTM baculovirus DNA”
  • the plate is then incubated for 5 hours at 27°C. After 5 hours the transfection solution is removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The plate is put back into an incubator and cultivation is continued at 27°C for four days .
  • plaque assay After four days the supernatant is collected and a plaque assay is performed, according to known methods.
  • An agarose gel with "Blue Gal” (Life Technologies, Inc., Rockville, MD) is used to allow easy identification and isolation of gal-expressing clones, which produce blue- stained plaques.
  • a detailed description of a "plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies, Inc., Rockville, MD, page 9-10). After appropriate incubation, blue stained plaques are picked with a micropipettor tip (e.g., Eppendorf) .
  • a micropipettor tip e.g., Eppendorf
  • the agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 ⁇ l of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4°C.
  • the recombinant virus is called V-hCRSPl .
  • Sf9 cells are grown in Grace's medium supplemented with 10% heat- inactivated FBS .
  • the cells are infected with the recombinant baculovirus V-hCRSPl at a multiplicity of infection ("MOI") of about 2.
  • MOI multiplicity of infection
  • the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available, e.g., from Life Technologies, Inc., Rockville, MD) . If radiolabeled polypeptides are desired, 42 hours later, 5 mCi of 35S- methionine and 5 mCi 35S-cysteine (available from Amersham) are added.
  • the cells are further incubated for 16 hours and then they are harvested by centrifugation.
  • the polypeptides in the supernatant as well as the intracellular polypeptides are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled) . Microsequencing of the amino acid sequence of the amino terminus of purified polypeptide can be used to determine the amino terminal sequence of the mature polypeptide and thus the cleavage point and length of the secretory signal peptide.
  • Exa ple 3 Cloning and Expression of hCRSPl in. Mammalian
  • a typical mammalian expression vector contains at least one promoter element, which mediates the initiation of transcription of mRNA, the polypeptide coding sequence, and signals required for the termination of transcr/iption and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV, HTLVI , HIVI and the early promoter of the cytomegalovirus (CMV) . However, cellular elements can also be used (e.g., the human actin promoter) .
  • Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, CA) , pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or pcDNA3.1/Hygro (+/-) (Invitrogen) , PSVL and PMSG (Pharmacia, Uppsala, Sweden) , pRSVcat (ATCC 37152) , pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109) .
  • vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, CA) , pcDNA3.1 (+/-), pcDNA/Zeo (+
  • Mammalian host cells that could be used include human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
  • the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome.
  • a selectable marker such as dhfr, gpt, neomycin, or hygromycin allows the identification and isolation of the transfected cells.
  • the transfected gene can also be amplified to express large amounts of the encoded polypeptide.
  • the DHFR (dihydrofolate reductase) marker is useful to develop cell lines that carry several hundred or even several thousand copies of the gene of interest.
  • Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy, et al., Biochem. J. 227:277-279 (1991); Bebbington, et al . , Bio/Technology 10:169-175 (1992)). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of polypeptides .
  • the expression vectors pCl and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen, et al . , Molec. Cell. Biol. 5:438-447 (1985)) plus a fragment of the CMV-enhancer (Boshart, et al . , Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, Xbal and Asp718, facilitate the cloning of the gene of interest.
  • the vectors contain in addition the 3 ' intron, the polyadenylation and termination signal of the rat preproinsulin gene.
  • the expression plasmid, phCRSPl HA is made by cloning a cDNA encoding hCRSPl into the expression vector pcDNAI/Amp or pcDNAIII (which can be obtained from Invitrogen, Inc.).
  • the expression vector pcDNAI/amp contains: (1) an E. coli origin of replication effective for propagation in E.
  • coli and other prokaryotic cells (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eucaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron; (5) several codons encoding a hemagglutinin fragment (i.e., an "HA" tag to facilitate purification) or HIS tag (see, e.g, Ausubel, supra) followed by a termination codon and polyadenylation signal arranged so that a cDNA can be conveniently placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker.
  • HA hemagglutinin fragment
  • HIS tag see, e.g, Ausubel, supra
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin polypeptide described by Wilson, et al . , Cell 37:767-778 (1984) .
  • the fusion of the HA tag to the target polypeptide allows easy detection and recovery of the recombinant polypeptide with an antibody that recognizes the HA epitope.
  • pcDNAIII contains, in addition, the selectable neomycin marker .
  • a DNA fragment encoding the hCRSPl is cloned into the polylinker region of the vector so that recombinant polypeptide expression is directed by the CMV promoter.
  • the plasmid construction strategy is as follows.
  • the hCRSPl cDNA of the deposited clone is amplified using primers that contain convenient restriction sites, much as described above for construction of vectors for expression of hCRSPl in E. coli.
  • suitable primers include those based on the coding sequences presented in at least one of SEQ ID NOS:l, 2, 3, as they encode hCRSPl polypeptides as described herein.
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with suitable restriction enzyme (s) and then ligated.
  • the ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037), and the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis or other means for the presence of the hCRSPl-encoding fragment .
  • COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in Sambrook, et al . , Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989) . Cells are incubated under conditions for expression of hCRSPl by the vector.
  • hCRSPl-HA fusion polypeptide is detected by radiolabeling and immunoprecipitation, using methods described in, for example Harlow, et al . ,
  • Plasmid pC4 is used for the expression of hCRSPl polypeptide.
  • Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146) .
  • the plasmid contains the mouse DHFR gene under control of the SV40 early promoter.
  • Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (ahCRSPlha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate .
  • MTX methotrexate
  • a second gene is linked to the DHFR gene, it is usually co-amplified and over-expressed. It is known in the art that this approach can be used to develop cell lines carrying more than 1,000 copies of the amplified gene(s). Subsequently, when the methotrexate is withdrawn, cell lines are obtained which contain the amplified gene integrated into one or more chromosome ( s ) of the host cell.
  • Plasmid pC4 contains for expressing the gene of interest the strong promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen, et al . , Molec. Cell. Biol. 5:438-447 (1985)) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart, et al . , Cell 41:521-530 (1985)). Downstream of the promoter are BamHI, Xbal, and Asp718 restriction enzyme cleavage sites that allow integration of the genes. Behind these cloning sites the plasmid contains the 3 ' intron and polyadenylation site of the rat preproinsulin gene.
  • LTR long terminal repeat
  • CMV cytomegalovirus
  • high efficiency promoters can also be used for the expression, e.g., the human b-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI .
  • Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express the hCRSPl in a regulated way in mammalian cells (M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-5551 (1992)).
  • Other signals e.g., from the human growth hormone or globin genes can be used as well.
  • Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate .
  • the plasmid pC4 is digested with restriction enzymes and then dephosphorylated using calf intestinal phosphatase by procedures known in the art.
  • the vector is then isolated from a 1% agarose gel.
  • the DNA sequence encoding the complete hCRSPl polypeptide is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene.
  • Non- limiting examples include 5 ' and 3 ' primers having nucleotides corresponding or complementary to a portion of the coding sequence of an hCRSPl, e.g., as presented in at least one of SEQ ID N0S:1, 2, 3, according to known method steps.
  • the amplified fragment is digested with suitable endonucleases and then purified again on a 1% agarose gel.
  • the isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase.
  • E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC4 using, for instance, restriction enzyme analysis.
  • Chinese hamster ovary (CHO) cells lacking an active DHFR gene are used for transfection.
  • 5 ⁇ g of the expression plasmid pC4 is cotransfected with 0.5 ⁇ g of the plasmid pSV2-neo using lipofectin.
  • the plasmid pSV2neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418.
  • the cells are seeded in ahCRSPlha minus MEM supplemented with 1 ⁇ g/ml G418.
  • the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in ahCRSPlha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 ⁇ g/ml G418. After about 10- 14 days single clones are trypsinized and then seeded in 6- well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM) .
  • methotrexate 50 nM, 100 nM, 200 nM, 400 nM, 800 nM
  • Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 mM, 2 mM, 5 mM, 10 mM, 20 mM) . The same procedure is repeated until clones are obtained which grow at a concentration of 100 - 200 mM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reverse phase HPLC analysis.
  • Northern blot analysis is carried out to examine hCRSPl gene expression in human tissues, using methods described by, among others, Sambrook, et al . , cited above.
  • a cDNA probe containing the entire nucleotide sequence of an hCRSPl polypeptide (SEQ ID NOS:l) is labeled with 32 P using the
  • the probe is purified using a CHROMA SPIN-100TM column (Clontech Laboratories, Inc.), according to the manufacturer's protocol number PT1200-1. The purified and labeled probe is used to examine various human tissues for hCRSPl mRNA.
  • MTN Multiple Tissue Northern
  • H human tissues
  • IM human immune system tissues
  • PCR polymerase chain reaction
  • nucleic acids see, e.g., Ausubel, supra, section 15
  • PCR polymerase chain reaction
  • nucleic acids e.g., SEQ ID N0:1, 2, 3, or any sequence described herein, as presented herein, to provide an hCRSPl polypeptide sequence of interest including at least one substitution, insertion or deletion selected from the group consisting of 66Y, 75A, 89K, 118S, 199F, 120T, 126T, 137D, 139K, 144S, 145S, 147Q, 148N, 152V, 154S, 156P, 158T, 1590, 161P, 162L, 163S, 164Q, 165G, 166N, 167S, 169R, 170K, 228R, 230N, 231
  • Unit 8.5 of Ausubel, supra contains two basic protocols for introducing base changes into specific DNA sequences.
  • Basic Protocol 1 as presented in the first section 8.5 of Ausubel, supra (entirely incorporated herein by reference) , describes the incorporation of a restriction site and Basic Protocol 2, as presented below and in the second section of Unit 8.5 of Ausubel, supra, details the generation of specific point mutations (all of the following references in this example are to sections of Ausubel et al . , eds., Current Protocols in Molecular Biology, Wiley Interscience, New York (1987-1999)).
  • An alternate protocol describes generating point mutations by sequential PCR steps. Although the general procedure is the same in all three protocols, there are differences in the design of the synthetic oligonucleotide primers and in the subsequent cloning and analyses of the amplified fragments.
  • the PCR procedure described here can rapidly, efficiently, and/or reproducibly introduce any desired change into a DNA fragment. It is similar to the oligonucleotide-directed mutagenesis method described in UNIT 8.1, but does not require the preparation of a uracil- substituted DNA template.
  • the main disadvantage of PCR-generated mutagenesis is related to the fidelity of the Taq DNA polymerase.
  • the mutation frequency for Taq DNA polymerase was initially estimated to be as high as 1/5000 per cycle (Saiki et al . , 1988) . This means that the entire amplified fragment must be sequenced to be sure that there are no Taq-derived mutations. To reduce the amount of sequencing required, it is best to introduce the mutation by amplifying as small a fragment as possible. With rapid and reproducible methods of double-stranded DNA sequencing (UNIT 7.4), the entire amplified fragment can usually be sequenced from a single primer.
  • DNA sample to be mutagenized Klenow fragment of E. coli DNA polymerase I (UNIT 3.5 of Ausubel, supra).
  • Appropriate restriction endonuclease (Table 8.5.1) Additional reagents and equipment for synthesis and purification of oligonucleotides (UNITS 2.11 & 2.12), phosphorylation of oligonucleotides (UNIT 3.10), electrophoresis of DNA on nondenaturing agarose and low gelling/melting agarose gels (UNITS 2.5A & 2.6), restriction endonuclease digestion (UNIT 3.1), ligation of DNA fragments (UNIT 3.16), transformation of E. coli (UNIT 1.8), plasmid DNA miniprep (UNIT 1.6), and DNA sequence analysis (UNIT 7.4)
  • oligonucleotide primers Prepare template DNA (see Basic Protocol 1, steps 1 and 2). Synthesize (UNIT 2.11) and purify (UNIT 2.12) the oligonucleotide primers (primers 3 and 4 in Fig. 8.5.2B). The oligonucleotide primers must be homologous to the template DNA for more than 15 bases. No four-base "clamp" sequence is added to these primers.
  • the primer sequences are based on a DNA encoding the hCRSPl polypeptide sequence of interest including at least one substitution, insertion or deletion selected from the group consisting of 66Y, 75A, 89K, 118S, 199F, 120T, 126T, 137D, 139K, 144S, 145S, 147Q, 148N, 152V, 154S, 156P, 158T, 1590, 161P, 162L, 163S, 164Q, 165G, 166N, 167S, 169R, 170K, 228R, 230N, 231P, 232R, 233E, 234G, 2351, 242P, 253Q, 269L, 277V, 278M, 280S, 282E, 283S, 284D, 302P, 306E, 307G, 310M, 3131, 316L, 321T, 351P, 355Q, 399L, 400N, 40
  • Phosphorylate the 5' end of the oligonucleotides (UNIT 3.10) . This step is necessary because the 5' end of the oligonucleotide will be used directly in cloning.
  • Amplify DNA and prepare blunt-end fragments Amplify the template DNA (see Basic Protocol 1, steps 5 and 6) .
  • After the final extension step add 5 U Klenow fragment to the reaction mix and incubate 15 min at 30°C.
  • the Taq polymerase adds an extra nontemplated nucleotide to the 3' end of the fragment. The 3 '-5' exonuclease activity of the Klenow fragment is required to make the ends flush and suitable for blunt-end cloning (UNIT 3.5).
  • ALTERNATE PROTOCOL INTRODUCTION OF A POINT MUTATION BY SEQUENTIAL PCR STEPS
  • the two fragments encompassing the mutation are annealed with each other and extended by mutually primed synthesis; this fragment is then amplified by a second PCR step, thereby avoiding the blunt-end ligation required in Basic Protocol 2.
  • This strategy is outlined in Figure 8.5.3. For materials, see Basic Protocols 1 and 2 of Ausubel, supra.
  • the oligonucleotides must be homologous to the template for 15 to 20 bases and must overlap with one another by at least 10 bases. The 5' end does not have a "clamp" sequence. Amplify the template DNA and generate blunt-end fragments (see Basic Protocol 2, steps 4 and 5) . Purify the fragments by nondenaturing agarose gel electrophoresis (UNIT).
  • each flanking sequence primer (each 1 uM final)
  • hCRSPl polypeptides In order to evaluate the proliferative effects of hCRSPl polynucleotides, hCRSPl polypeptides, hCRSPl agonists, hCRSPl antagonists, and hCRSPl antibodies gross changes in the number of cells remaining in culture after exposure to a particular test agent for a period of three days are measured. Prior to assay, cells derived from various cell lines including, but not limited to, those shown below, are incubated in an appropriate assay medium to produce a sub-optimal growth rate, e.g., a 1:10 or 1:20 dilution of normal culture medium.
  • a sub-optimal growth rate e.g., a 1:10 or 1:20 dilution of normal culture medium.
  • Cells are grown in T-150 flasks, then harvested by trypsin digestion and replated at 40 to 50% confluence into poly-D-lysine-treated 96-well plates. Cells are only plated into the inner 32 wells to prevent edge artifacts due to medium evaporation; the outer wells are filled with buffer alone. Following incubation overnight to stabilize cell recovery, a test agent is added to the appropriate wells. Each test agent is assayed in triplicate at two different concentrations, IX and 0. IX dilution in assay medium. Two controls are also included on each assay plate: assay medium and normal growth medium. After approximately 72 hours of exposure, the plates are processed to determine the number of viable cells. Plates are spun to increase the attachment of cells to the plate.
  • the medium is then discarded, and 50 ⁇ L of detection buffer is added to each well.
  • the detection buffer consisted on MEM medium containing no phenol red (Gibco) with calcein AM (Molecular Probes) and PLURONICTM F-127 (Molecular Probes) , each at a 1:2000 dilution. After incubating the plates in the dark at room temperature for thirty minutes, the fluorescence intensity of each well is measured using a Cytofluor 4000-plate reader (PerSeptive Biosystems) . For a given cell type, the larger the fluorescence intensity, the greater the number of cells in the well .
  • GT1-7 hypothalamic SV40 transformed cell line
  • SK-N-MC ATCC HTB-10
  • HEP-G2 (ATCC HB-8065)
  • ECV304 endothelial cell line
  • GLUTag SV40 Tag transformed enteroendocrine cell line
  • HUVEC (Clonetics # CC2517T150)
  • balb/c 3T3 (ATCC CCL-163) HDF (dermal fibroblasts) (Clonetics # CC2511T150)
  • M07E leukemia cell line
  • THP-1 (ATCC TIB-202)
  • Binding of hCRSPl proteins to human tissues can be determined by protein staining with fluorescent dye. All tissues are fixed with 3% paraformaldehyde and embedded in paraffin. Tissues are prepared for analysis by removing the paraffin with xylene then gradually rehydrating the tissue with graded solutions of ethanol and water. Antigen retrieval is performed to unmask antigenic sites so that antibodies can recognize the antigen. This is accomplished by soaking the tissue in citrate buffer (Dako, Carpinteria, CA) for twenty minutes at 80 to 90°C followed 10 minutes at ambient temperature. The tissue is then washed in tris- buffered saline (TBS) containing 0.05% TWEENTM 20 and 0.01% thimerosol . To minimize non-specific background staining, the tissue is soaked in non-serum protein block (Dako) for 45 minutes, after which the protein block is removed by blowing air over the tissue.
  • Dako non-serum protein block
  • the tissue is exposed for 2 hours to the FLAG-HIS tagged hCRSPl protein at 10 ⁇ g/mL. Following exposure, the tissue is washed twice with tris-buffered saline (TBS) containing 0.05% TWEENTM 20 and 0.01% thimerosol. The tissue sample is then incubated for one hour with mouse anti-FLAG antibody at 10 ⁇ g/mL. Subsequently, the tissue is washed twice with tris-buffered saline (TBS) containing
  • TWEENTM 20 and 0.01% thimerosol 0.05% TWEENTM 20 and 0.01% thimerosol.
  • the tissue is exposed to rabbit anti-mouse Ig with Alexa 568, a fluorescent dye, at 10 ⁇ g/mL for one hour, followed again by two washes with tris-buffered saline (TBS) containing 0.05% TWEENTM 20 and 0.01% thimerosol. Finally, the tissue is coverslipped with fluorescence mounting media, and the fluorescence is measured.
  • TBS tris-buffered saline

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne au moins un nouveau polypeptide hCRSP1 et des acides nucléiques isolés qui codent ce ou ces polypeptides hCRSP1, des polypeptides hCRSP1, des vecteurs, des cellules hôtes, des produits transgéniques, des chimères et des procédés de préparation et d'utilisation de ces derniers ainsi que des anticorps à spécificité hCRSP1 et des procédés associés.
PCT/US2001/003685 2000-03-06 2001-02-21 Acides nucleiques codant des polypeptides crsp1 humains et utilisations de ces derniers WO2001066738A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001239742A AU2001239742A1 (en) 2000-03-06 2001-02-21 Nucleic acids, encoding human crsp1 and uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US18711700P 2000-03-06 2000-03-06
US60/187,117 2000-03-06

Publications (2)

Publication Number Publication Date
WO2001066738A2 true WO2001066738A2 (fr) 2001-09-13
WO2001066738A3 WO2001066738A3 (fr) 2002-03-07

Family

ID=22687664

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/003685 WO2001066738A2 (fr) 2000-03-06 2001-02-21 Acides nucleiques codant des polypeptides crsp1 humains et utilisations de ces derniers

Country Status (2)

Country Link
AU (1) AU2001239742A1 (fr)
WO (1) WO2001066738A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1511506A4 (fr) * 2002-06-13 2006-12-06 Metabolex Inc Compositions et methodes permettant de prevenir, de traiter et de diagnostiquer des diabetes

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020015950A1 (en) * 1999-07-07 2002-02-07 Karen Anne Jones Atherosclerosis-associated genes
EP1444260A4 (fr) * 1999-05-14 2004-08-25 Millennium Pharm Inc Proteines secretees et leur utilisation

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1511506A4 (fr) * 2002-06-13 2006-12-06 Metabolex Inc Compositions et methodes permettant de prevenir, de traiter et de diagnostiquer des diabetes

Also Published As

Publication number Publication date
WO2001066738A3 (fr) 2002-03-07
AU2001239742A1 (en) 2001-09-17

Similar Documents

Publication Publication Date Title
US7579180B2 (en) Beta secretase polypeptides
EP1303605A2 (fr) Acides nucleiques, vecteurs, cellules hotes, polypeptides et leurs utilisations
JP2011155971A (ja) Ｉｌ−１関連ポリペプチド
CA2706859A1 (fr) Facteur de croissance transformant beta - 9 mammaliens
WO2000059942A2 (fr) COMPOSITIONS hOB-BP2h, LEURS TECHNIQUES ET LEURS UTILISATIONS
WO2002002771A2 (fr) Nouveau gene hsparc-11 et procedes de fabrication et d'utilisation de ce dernier
WO2001092522A2 (fr) Acides nucleiques et polypeptides humains fgf-20
WO2000015781A1 (fr) Antagonistes du facteur de croissance des fibroblastes
JP2002508166A (ja) ヒトＤｅｎｄｒｉａｃおよびＢｒａｉｎｉａｃ−３
WO2002026801A2 (fr) Nouvelles proteines secretees et utilisations de ces dernieres
US20030211991A1 (en) Human sez6 nucleic acids and polypeptides
WO2001066738A2 (fr) Acides nucleiques codant des polypeptides crsp1 humains et utilisations de ces derniers
US20050014222A1 (en) Phosphatases which activate map kinase pathways
CA2341688A1 (fr) Acides nucleiques d'homologue du facteur 8, polypeptides, procedes et utilisations
WO2001083552A2 (fr) Acides nucleiques et polypeptides sez6 humains
EP1313765A2 (fr) Nouvelles proteines secretees et procedes d'utilisation de ces dernieres
WO2000005248A1 (fr) Cellules hotes, vecteurs, polypeptides, acides nucleiques de gdf-3 humains et leur methodes d'utilisation
WO2001000662A2 (fr) Utilisation de polypeptides fgh-8 humains comme agents neurotrophiques
WO2000006714A1 (fr) ACIDES NUCLEIQUES DDKh-3, POLYPEPTIDES, VECTEURS, CELLULES HOTES, PROCEDES ET UTILISATIONS
WO2000012117A1 (fr) ACIDES NUCLEIQUES, POLYPEPTIDES HOMOLOGUES DE Wnt, PROCEDES ET UTILISATIONS
WO2000037492A2 (fr) ACIDES NUCLEIQUES huHDGFH, POLYPEPTIDES, PROCEDES ET UTILISATIONS DE CEUX-CI
WO2000034782A1 (fr) Compositions flint et leurs utilisations
US6800731B2 (en) Human oncogene induced secreted protein I
US20030120039A1 (en) Human preoptic regulatory factor-2 and uses thereof
WO2001042464A2 (fr) Facteur 2 de regulation preoptique humain et ses utilisations

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载