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WO1990006369A1 - Utilisation d'un site de clivage de protease de vih pour exprimer des proteines matures - Google Patents

Utilisation d'un site de clivage de protease de vih pour exprimer des proteines matures Download PDF

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Publication number
WO1990006369A1
WO1990006369A1 PCT/US1989/005009 US8905009W WO9006369A1 WO 1990006369 A1 WO1990006369 A1 WO 1990006369A1 US 8905009 W US8905009 W US 8905009W WO 9006369 A1 WO9006369 A1 WO 9006369A1
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Prior art keywords
protease
cleavage site
nucleotide sequence
fusion protein
sequence
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PCT/US1989/005009
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English (en)
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Christine Marie Debouck
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Smithkline Beecham Corporation
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Publication of WO1990006369A1 publication Critical patent/WO1990006369A1/fr

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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses
    • C12N9/506—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses derived from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • This invention relates to the field of recombinant DNA techniques for production of proteins and polypeptides.
  • Fusion proteins can be cleaved by chemical means, such as cyanogen bromide, or more specific cleavage of hybrid proteins can be achieved by including a nucleotide sequence encoding a protease cleavage site or other selectively cleavable site into the nucleotide sequence encoding the hybrid protein between the sequences encoding the respective proteins.
  • chemical means such as cyanogen bromide
  • cleavage of hybrid proteins can be achieved by including a nucleotide sequence encoding a protease cleavage site or other selectively cleavable site into the nucleotide sequence encoding the hybrid protein between the sequences encoding the respective proteins.
  • Ile-Glu-Gly-Arg is the sequence that immediately precedes the two factor X a cleavage sites in prothrombin.
  • the purified hybrid protein was harvested and subsequently digested with blood coagulation factor X a , which had been activated with
  • Russel's viper venom Russel's viper venom.
  • Germino and Bastia Proc. Nat'l Acad. Sci., USA, 81: 4692-4696 (1984) described a similar system using an initiator sequence, a sequence encoding 60 amino acids of chicken pro-alpha-2 collagen and a marker gene.
  • the resulting hybrid protein was purified and subsequently cleaved by collagenase.
  • This system had the drawback of apparently non-specific proteolysis when high concentration of collagenase were used to cleave the hybrid protein, and it was necessary to carefully titrate each batch of collagenase to ensure specific cleavage and to guard against this apparently non-specific proteolysis.
  • Patent 4,751,180 issued June 14, 1988 to Cousens et al discloses methods and compositions for producing heterologous proteins in a host organism such as yeast.
  • a DNA sequence coding for superoxide dismutase or other highly expressed protein is joined to a DNA sequen coding for a polypeptide of interest by a selectively cleavable linkage.
  • the fusion protein thus produced can then be purified and subsequently cleaved by chemical or enzymatic means to produce the polypeptide of interest.
  • proteases e.g., trypsin or cyanogen bromide is relatively non-specific. As a result, functional proteins are difficult to produce with these methods. In the case of site-specific proteases, cleavage requires an extra processing step following production of a fusion protein. Poor solubility of the fusion proteins in the cells that produce them is also often a drawback of these methods.
  • the invention provides methods of preparing a protein product by expressing, in the same cell, a fusion protein and a protease capable of cleaving the protease cleavage site, contained in the fusion protein, to
  • HIV-1 protease cleavage of a fusion protein having and HIV-1 protease cleavage site to produce the selected protein product is more selective than when cleavage is done with chemical reagents or enzymes such as trypsin.
  • the amino acid sequence recognized by these latter methods may appear several times in the fusion protein, and the use of these methods will result in several, usually non-fictional, protein fragments.
  • the retroviral cleavage site is not commonly found in proteins, thus, the likelihood of inadvertently cleaving the protein product while cleaving the deliberate retroviral protease cleavage site is correspondingly significantly reduced.
  • the invention provides the efficient production of protein products in quantities sufficient for
  • the invention eliminates the need to first purify the fusion protein, release the protein product and then isolate the protein product in a second, separate purification process.
  • the protein product is present in cells in a mature form. There should be little or no cost for large quantities of purified protease to cleave the fusion protein, since the organism is producing the appropriate enzyme at the same time it is producing the fusion protein.
  • the methods of the invention will result in more efficient, less
  • a first recombinant DNA molecule comprising a fusion protein nucleotide sequence coding for a fusion protein is provided.
  • the fusion protean nucleotide sequence is provided.
  • the first portion preferably codes for a protein that is
  • the protease cleavage site portion codes for amino acids recognized by the protease selected for use in the invention.
  • the protein product portion codes for a selected protein product.
  • the amino acids of the cleavage site act as substrate for the proteolytic enzyme. Cleavage of the protease recognition site releases the protein product from its association with the first portion of the fusion protein.
  • a second recombinant DNA molecule comprising a coding sequence for a protease or an active fragment or derivative thereof, capable of cleaving amino acids encoded by the protease cleavage site portion is also provided.
  • This nucleotide sequence codes for a functional protease that enzymatically releases the protein product from the fusion protein.
  • the first and second recombinant DNA molecules are then introduced into a cell for expression of the nucleotide sequences and production of the protein
  • the cells are cultured under conditions selected to express the fusion protein and the protease, whereby the protease cleaves the fusion protein at the protease cleavage site to thereby produce the mature protein product.
  • the mature protein product may then be isolated by conventional methods.
  • the fusion protein nucleotide sequence and the protease nucleotide sequence may be incorporated into at least one expression vector and then introduced into the cell, or the two sequences may be incorporated into the genome of the cell for expression of the fusion protein and protease.
  • the first and second recombinant DNA molecules are
  • the protease and cleavage site are preferably derived from HIV-1.
  • the first and second recombinant DNA molecules may further comprise at least one expression control sequence operatively linked with the fusion protein nucleotide sequence and the protease nucleotide sequence, respectively.
  • the invention also provides fusion protein nucleotide sequences as described above, nucleotide sequences coding for HIV-1 protease or proteolytically active fragments or derivatives thereof, recombinant
  • the invention further provides cells having incorporated therein two recombinant DNA molecules, a first recombinant DNA molecule comprising a nucleotide sequence coding for a fusion protein nucleotide sequence and a second recombinant DNA molecule comprising a nucleotide sequence coding for a protease capable of cleaving the fusion protein at the protease cleavage site.
  • the fusion protein nucleotide sequences are those described herein for the production of fusion proteins.
  • the cells of the invention preferably have two expression vectors, one of which comprises the first recombinant DNA molecule, and the other of which comprises the second recombinant DNA molecule.
  • the invention provides nucleotide sequences, peptides, cells and methods for producing protein
  • a retroviral protease is coupled with a retroviral protease cleavage site for production of mature protein products.
  • Preferred retroviruses are the immunodeficiency viruses; more preferably, Human Immunodeficiency Virus-1 (HIV-1).
  • Retroviruses that is, viruses within the family Retroviridae, are a large family of enveloped, icosohedral viruses of about 150 nm having a coiled nucleocapsid within the core structure and having RNA as the genetic material.
  • the family comprises the oncoviruses, such as the sarcoma and leukemia viruses, the immunodeficiency viruses and the lentiviruses.
  • HIV causative agent of acquired immunodeficiency syndrome and related disorders
  • Retroviridae family There exist several isolates of HIV, including human T-lymphotropic virus type-Ill (HTLV-III). the lymphadenopathy virus (LAV) and the AIDS-associated retrovirus (ARV) which have been grouped in type 1.
  • HTLV-III human T-lymphotropic virus type-Ill
  • LAV lymphadenopathy virus
  • ARV AIDS-associated retrovirus
  • HIV type 2 which has been shown to be associated with AIDS in West Africa.
  • Immunodeficiency Virus such as SIV mac -BK28 [see
  • HIV genome Molecular characterization of the HIV genome has demonstrated that the virus exhibits the same overall gag-pol-env genetic organization as other retroviruses.
  • HIV also contains a highly specific protease responsible for post-translational processing of HIV precursor
  • gag-pol region is initially translated int a polyprotein precursor of about 55 kDa that is then processed into the mature p17, p24, and p15 gag structural proteins. Similarly, gag-pol region is believed to be translated into a larger precursor through a translational frameshift between the overlapping gag and pol reading frames. This gag-pol precursor is post-translationally processed as well, to yield the mature gag proteins and the products of the pol region , including protease ,
  • gag and gag-pol polyproteins maturation of the gag and gag-pol polyproteins has been shown to be effected at least, in part, by a
  • the retrovirus protease is essential to the retroviral life cycle as indicated by the roduction of noninfectious, replication-deficient virions by Moloney murine leukemia virus variants mutated in the protease coding region.
  • HIV protease codin ion is believed to be positioned between the p15 gag and reverse transcriptase genes and to reside entirely with the pol reading frame. Determination of the amino-terminal sequences of HIV gag p24 and reverse transcriptase proteins and knowledge of the surrounding sequences have revealed a common sequence [Asn-(Phe or Tyr)*Pro-Ile] at the cleavage site, with cleavage occurring at the asterisk between (phenylalanine or tyrosine) and proline. A similar sequence,
  • the first and second recombinant DNA sequences are generally introduced into a cell for production of the protein product either by incorporation into at least one expression vector that is then introduced into the cell, or. by integration into the genome of the host cell.
  • nucleic acid sequences of the invention will be prepared as a single entity, it should be appreciated that they may be prepared as various fragments, these fragments joined in expression cassettes and expression vectors to various untranslated regions providing for particular functions , and ultimately the coding sequences brought together at a subsequent stage.
  • the discussion will be directed primarily to the situation where the coding sequence is prepared as a single entity and then transferred to an expression vector.
  • the fusion protein nucleic acid sequences of the invention preferably comprise a first portion, a protease cleavage site portion and a protein product portion.
  • the parts of the fusion protein nucleic acid sequence are preferably arranged in an order wherein the first portion is upstream of the protease cleavage site portion and the protein product portion, respectively.
  • the protein product portion may be upstream of the protease cleavage site portion and the first portion.
  • the first portion of the nucleotide sequences of the invention are preferably chosen from genes for
  • nucleic acid sequences encoding proteins produced in amounts of about 5% or greater of the total protein produced by the cell are considered to be
  • nucleic acid sequence for use in the invention may be identical to the gene encoding the well-expressed protein portion, or it may be a mutant of the gene or have one or more codons substituted.
  • the entire gene or any portion of the gene which provides for the desired high yield of protein in the host may be employed.
  • nucleotide sequence when the nucleotide sequence is to be expressed in E. coli, several genes, such as the galactokinase gene, Rosenberg et al, Meth. Enzymol.,
  • a preferred first portion is an E. coli galactokinase gene, more preferably the first 56 codons of the E. coli galactokinase gene. This gene and gene fragment result in high levels of expression in E. coli.
  • the choice of protein product portion will depend upon the identity of the selected protein product.
  • the protein products of the invention are mature proteins or polypeptides and do not comprise generally amino acids derived from the first portion.
  • the protein products may however, comprise one or more amino acids derived from the protease cleavage site portion, depending on the length of the protease cleavage site portion and the exact position of cleavage within this portion.
  • the protein product portion can code for an entire protein, part of a protein polypeptide, or oligopeptide. The size and choice of protein product will depend on such factors as its use or desired characteristics. Protein products of interest include enzymes, hormones, blood clotting factors,
  • immunoglobulins etc., as well as fragments or parts of such molecules.
  • the protease cleavage site portion comprises nucleotides coding for the amino acids forming the
  • protease cleavage site portion should be chosen to provide an enzyme-substrate pair with the protease nucleotide sequence. For example, if the protease cleaves an Ala-Leu bond, the protease cleavage site portion should include at least nucleotides coding these amino acids. Suitable protease cleavage sites for use in the invention include those which serve as substrates for proteases from
  • protease cleavage site preferably
  • Suitable proteases for use in the invention include those that are capable of exhibiting proteolytic activity within the environment of the cell in which the expression system is introduced and that are not
  • proteases substantially toxic for the host cell upon induction of the expression system.
  • Proteolytically active fragments and derivatives of such proteases are also suitable for use in the invention.
  • Proteases, such as trypsin, having cleavage site which commonly, and frequently, appear in protein products are not suitable for use in the invention.
  • these proteases may, nevertheless, be suitable for use in the invention in situation where the protein product is functional after cleavage of the fusion protein with the protease.
  • Preferred protease cleavage sites include those of retroviruses, preferably immunodeficiency virus
  • cleavage sites such as those of HIV and Simian
  • Preferred cleavage sites are selected from the group consisting of R 1 .tyrosine.proline.R 2 ,
  • R 2 and R 1 . leucine. phenylalanine. R 2 wherein R 1 is
  • X 1 , X 2 , X 3 , X 4 and X 5 are independently an
  • n,o,p,r and s are independently 0 or 1.
  • X 1 is preferably selected from the group consisting of serine and threonine.
  • X 1 , X 2 , X 3 , X 4 and X 5 may
  • X 4 and X 5 are preferably hydrophobic amino
  • acids such as isoleucine, valine, glycine or serine.
  • cleavage sites are:
  • amino acids are those conventionally given to amino acids found in
  • proteins i.e., ala-alanine, arg-arginine, asp-aspartic acid, asn-asparagine, cys-cysteine, gly-glycine,
  • glu-glutamic acid gln-glycine, his-histadine,
  • the protease cleavage site portion may also include additional nucleotides flanking the nucleotides coding for the cleavage site, providing these additional nucleotides do not encode amino acids that substantially interfere with the activity of the protease or the
  • nucleotide sequences of the invention are those coding for further amino acids near the protease cleavage site, those derived from the genetic material of the organism from which the protease cleavage site was isolated, linker sequences, or sequences tending to promote proper folding of the fusion protein produced from the nucleotide sequences of the invention.
  • a nucleotide sequence coding for HIV-1 protease may be obtained by restriction endonuclease digestion of clone of HIV-1, such as the BH10 clone of the HTLVIIIB isolate of HIV, by isolating HIV-1 from persons infected with the virus, and synthesis of single-stranded DNA from the HIV RNA with reverse transcriptase using conventiona techniques. The single-stranded DNA complementary may then be used as the template for preparing a second strand to provide double-stranded cDNA containing the coding region for the HIV-1 protease. Once DNA encoding of the protease region is obtained, fragments containing the nucleotide sequence coding for protease are obtained by digestion of the DNA with appropriate restriction
  • fragment containing the nucleotide sequence coding for protease is isolated in accordance with conventional techniques. Fragments, mutations, additions and deletions from the nucleotide sequence coding for HIV-1 protease that have proteolytic activity are also within the scope of the invention and are suitable for use in the methods of the invention.
  • the DNA sequences coding for the first portion, the protease cleavage site portion, the protein product portion, and the DNA sequence coding for the protease may be obtained in a variety of ways.
  • the sequences encoding the portions may be derived from natural sources, where the messenger RNA or chromosomal DNA may be identified with appropriate probes, which are complementary to a portion of the coding or non-coding sequence. From
  • messenger RNA, single-stranded (ss) DNA may be prepared employing reverse transcriptase in accordance with
  • the ss DNA complementary strand may then be used as the template for preparing a second strand to provide double-stranded (ds) cDNA containing the coding region for the portion.
  • ds double-stranded
  • the region containing the coding region may be detected employing probes, restriction mapped, and by appropriate techniques isolated, substantially free of untranslated 5' and 3' regions.
  • the remaining portions may be provided by synthesis of adapters which can be ligated to the coding portions and provide for convenient termini for ligation to other sequences providing
  • the first portion, the protein product portion, the protease cleavage site portion, and the DNA sequence coding for the protease may also be prepared synthetically using conventional methods and reagents for synthesizing nucleic acids.
  • the first and second recombinant DNA molecules may further comprise expression control elements
  • the fusion protein nucleotide sequence is preferably flanked by expression control elements that provide control of transcription and translation. These may be included with the fusion protein nucleotide
  • the fusion protein nucleic acid sequence in the first recombinant DNA molecule to form an expression cassette can be moved intact into a selected expression vector.
  • the fusion protein nucleic acid sequence, without transcription control elements can be moved to selected expression vectors already having transcription control elements incorporated therein.
  • An expression cassette containing the nucleic acid sequences of the invention preferably comprises, in an upstream position, a promoter, followed by a
  • translation initiation signal comprising a ribosome binding site, and an initiation codon, and, in a
  • transcription termination signal downstream position, a transcription termination signal.
  • additional transcription control elements such as capping sequences and enhancers may also be used when expression is done in eukaryotic hosts.
  • the transcription and translation control elements may be ligated in any functional combination or order.
  • the transcription and translation control elements used in any particular embodiment of the invention will be chosen with reference to the type of cell into which the expression vector will be introduced, so that an expression system will be created.
  • Nucleotide sequences coding for the protease also preferably form parts of an expression cassette containing the elements described above for the nucleotide sequences coding for the fusion protein, or they may be inserted into expression vectors already containing transcription control elements. If the nucleotide sequences coding fo r the fusion protein and the protease are directed by different sets of transcription control elements, it is preferable that both sequences are expressed in comparable amounts to avoid a relative overabundance of the fusion protein or protease.
  • inducible promoters are preferred for use in the invention.
  • suitable inducible promoters are those where induction may be as a result of a physical change, e.g., temperature; or chemical change, e.g., change in nutrient or metabolite concentration, such as glucose or tryptophan; or change in pH or ionic strength.
  • Suitable promoters in E. coli include promoters, such as the Lambda P L and P R promoters, and the E. coli
  • MMTV mouse mammalian tumor virus
  • the transcription initiation elements can be separated from the transcription terminator sequence by a polylinker, which has a plurality of unique restriction sites for insertion of the fusion protein nucleic acid sequence.
  • the polylinker will be followed by the
  • the polylinker will be cleaved and the open reading frame nucleotide sequence coding for the fusion protein may be inserted.
  • the expression vector will be selected so as to have an appropriate copy number, as well as providing for stable extrachromosomal maintenance.
  • the expression vector will usually have a marker that allows for
  • Suitable markers include those which provide the host with an enhanced ability to utilize an essential nutrient or metabolite in short supply, or provide resistance to drugs or
  • antibiotics such as ampicillin or chloramphenicol.
  • Cells suitable for use in the invention include prokaryotic and eukaryotic cells that can be transformed to stably contain and express both the fusion protein and the protease. Suitable types of cells include bacterial, yeast and mammalian cells. E. coli cells are preferred. Introduction of at least one expression vector
  • incorporating the fusion protein nucleotide sequence and the protease nucleotide sequence can be performed in a variety of ways, such as calcium chloride or lithium chloride treatment, calcium-polyethylene glycol-treated DNA with spheroplasts, use of liposomes, mating,
  • the plasmid When one plasmid is employed for expression of both the fusion protein and the protease, the plasmid may be constructed to have two separate sets of transcription control elements, one for the fusion protein nucleic acid sequence and one for the protease nucleotide sequence; or both the fusion protein and the protease may be under the control of the same transcription control elements.
  • the transcription control elements When two plasmids are used, the transcription control elements may be selected to be identical in both plasmids, or the may be selected to be different in one or more
  • both are inserted into the same cell. Care should be taken that the expression vectors are chosen to be compatible and stable in the same cell.
  • E. coli cell are chosen for production of the protein product, and two plasmids are used (one for the fusion protein, one for the protease), a suitable compatible pair of plasmids is one plasmid carrying the colEl origin of replication and the ampicillin-resistance marker and the other plasmid
  • chloramphenicol-resistance marker Other suitable compounds
  • Saccharomyces cerevisiae (micron-based and CEN-based expression vectors), Gorman et al, Gene, 48:13-22 (1986). Stable expression of both the fusion protein and the protease using two expression vectors should also be possible with some mammalian cells.
  • Cells, expression vectors and transcription control elements are selected to be complementary to one another to form an expression system.
  • Suitable expressio systems in E. coli utilize, for example, influenza NSl, Young et al, Proc. Nat'l Acad. Sci., USA, 80:6105 (1983), phage Lambda O protein, Rosenberg et al, Meth. Enzymol., 101:123 (1983), the E. coli galactokinase protein,
  • yeast expression can be achieved with E. coli galK, Rosenberg et al, in
  • At least one expression cassette comprising the first and second recombinant DNA molecules can be
  • the expression cassette can be incorporated into the genome of the host cell by site-directed, e.g., by homologous recombination, or random integration. Introduction of the expression cassette into the host cell can be accomplished in. a variety of ways, such as co-precipitation, calcium chloride or lithium chloride treatment, calcium
  • nucleotide sequence coding for the fusion protein and the nucleotide sequence coding for the protease may be under the control of the same or different transcription control elements. Additionally, the nucleotide sequence coding for the fusion protein and the nucleotide sequence coding for the protease may be incorporated into the same cell in different manners, i.e., the first recombinant DNA molecule coding for the fusion protein may be incorporated into the genome of the host cell, and the second
  • sequence coding for the protease may be incorporated into an expression vector, or vice versa.
  • Host cells containing the first and second recombinant DNA molecules are then grown in an appropriate medium for the host.
  • an inducible promoter employed, the host cell may be grown to high density and the promoter turned on for expression of the fusion protein and protease. Where the promoter is not
  • the cells may be grown until there is no further increase in product formation or the ratio of nutrients consumed to product formation falls below a predetermine level, at which time, the cells may be harvested, lysed and the protein product obtained and purified in
  • E. coli The bacterium E. coli is the organism of choice for the production of the protein products. Cloning and expression can be obtained rapidly in E. coli; and to date, in some cases, much higher levels of gene expression have been achieved in E. coli as compared to other
  • E. coli organisms, e.g., mammalian systems (Chinese hamster ovary cells) or yeast. Production in E. coli is readily
  • E. coli strains are defective lysogen strains. Some such strains can be used in combination with the inducible Lambda phage P L promoter for heat induction of the promoter, Devare et al,
  • Nucleotide sequences coding for HIV-1 protease or a proteolytically active fragment or derivative thereof may be obtained by restriction endonuclease digestion of a clone of HIV-1, using the appropriate restriction
  • Proteolytically active fragments, derivatives, additions and deletions of nucleotide sequences coding for HIV-1 protease are also within the scope of the invention.
  • Suitable nucleotide sequences coding for peptides having proteolytic activity towards HIV-1 cleavage sites include, or the complementary sequences thereof:
  • the recombinant E. coli protease product is produced by recombinant DNA techniques from nucleotide sequences coding for nucleotide sequences coding for peptides having proteolytic activity toward HIV-1 cleava sites.
  • the recombinant E. coli protease has proteolytic activity towards HIV-1 cleavage sites and is useful for cleaving fusion proteins as disclosed herein. Suitable methods and nucleotide sequences for preparing the
  • E. coli protease product include those
  • the protease product may be produced in cells with or without production of a fusion protein.
  • Suitable peptides include those selected from the group of peptides consisting of-
  • Proteolytically active fragments, mutations, additions or deletions of the recombinant E. coli protease product are also within the scope of the invention.
  • the E. coli strain used for expression was AR120, as described in Mott et al, Proc. Nat'l Acad. Sci., USA, 82: 88-92 (1985). All DNA manipulations were carried out as described in Maniatis et al. Molecular Cloning: A
  • E. coli strain AR120 carrying the expression plasmids were induced, and total cell extracts were prepared and analyze by NaDodSO 4 /PAGE as described in Aldovini, Debouck et al, Proc. Nat'l Acad. Sci., USA, 83:6672-6676 (1886). HIV Protease and Activity
  • HIV-1 protease Vectors for expression of HIV-1 protease were constructe by inserting the appropriate restriction fragment into the expression vector pOTSKF33 All HIV-1 restriction fragments were isolated from the BH10 clone of the HTLVIIIB isolate of HIV (Shaw et al, Science, 226:1165-1171 (1984).
  • pOTSKF33 was constructed in several steps:
  • Step 1 Insertion of the polylinker region of pUC19 into the pOTS34 vector: pOTS34, a derivative of pASl (Devare et al, Cell, 36:43 (1984), was digested with the Bglll restriction endonuclease, treated with DNA polymerase I (Klenow) to create blunt ends and redigested with the Sphl restriction endonuclease.
  • the large 5251 base pair (bp) Bglll-Sphl fragment was purified and ligated to the 135 bp Bglll-Sp restriction fragment isolated and purified from pUC19 (Yanisch-Perron et al, Gene, 33:103 (1985).
  • Step 2 Insertion of the partial galK expression unit from pASK into pOTS-UC: pOTS-UC was digested with Xmal, treated with Klenow to create blunt ends and redigested with Hindlll. The large fragment was purified and ligated to a 2007 bp Bell filled-Hindlll fragment isolated and purified from pASK. The resulting plasmid was called pOTSKF45.
  • the pASK plasmid vector is a pASl derivative in which the entire galK coding sequence is inserted after the ATG initiation codon. This galK sequence differs from the authentic sequence (Debouck et al, Nucleic Acids Res., 13:1841 (1985)) at the 5' end:
  • the pASK sequence contains a BamHI site (GGATCC) at the ATG whereas the authentic sequence does not.
  • pOTSKF45 was digested with Xmal and Sail and then treated with Klenow to create blunt ends.
  • 5'-GTA.GGC.CTA.GTT.AAC.TAG-3' was then introduced between the Xmal filled and Sail filled sites by ligation with T4 DNA ligase to yield the vector: pOTSKF33.
  • NlalV-Hae III fragment was isolated from BH10 using standard cloning techniques and inserted at the Stul restriction site of pOTSKF33 in the correct orientation.
  • the NlalV-Haelll restriction fragment has the following nucleotide sequence and corresponding translation for the reading frame of interest:
  • a 516-bp Hae III fragment was isolated from BH 10, using standard cloning techniques and inserted at the XmaI filled-in restriction site of pOTSKF33 in the correct orientation.
  • the Hae III restriction fragment has the following nucleotide sequence and corresponding
  • E. coli overlapping fragments, encompassing the protease coding region, were inserted into the pOTSKF33 bacterial expression vector, as described above in I.
  • the fragments contained the two domains (I and II) that are highly conserved among all known retroviral protease, as well as the region between these domains.
  • the fragments differed only in the amount of viral sequence information they carried upstream and downstream from the conserved
  • the PRO3 and PRO4 fragments contained the proteolytic cleavage site, (indicated by the asterisk) Thr-Leu-Asn-Phe*Pro, positioned downstream of domain II, from which the amino terminus of reverse transcriptase derives ( Figure 1B) and another protease cleavage site, Ser-Phe-Asn-Phe*Pro, positioned approximately 20 codons upstream of conserved domain I, from which the amino terminus of HIV-1 protease derives. Proteolytic cleavage at these two sites yields a 10-kDa polypeptide which correspond to the mature protease.
  • the protein products expressed by the PRO4 constructs were examined initially by NaDodSO./PAGE by the method described in Aldovini, Debouck et al, supra.
  • the PRO3 and PRO44 constructs did not produce proteins with the size expected for their encoded products (20 kDa and 25 kDa, respectively). Instead, both
  • constructs gave rise to a protein of approximately 10 kDa that was visualized by immunoblot analysis using a
  • Plasmid PRO4-BX was derived from the PRO4 expression vector by digestion with Bel I, treatment with DNA polymerase (Klenow) and ligation with the 8-mer oligonucleotide 5' CCTCGAGG 3'. This treatment resulted in the insertion of four codons (encoding Pro-Ser-Arg-Asp) in the protease region between the conserved domains.
  • the pOTSKF33 bacterial expression vector As described above in I, was used. A translational fusion was created between the HIV-1 gag reading frame and the first 56 amino acids of galactokinase sequence on the expression vector. A 1286-base pair (bp) Cla I-Bg II filled-in restriction endonuclease fragment, including most of the pl7, all of the p24, and half of the p15 gag coding sequence, was inserted at the Stul site of the pOTSKF33 fusion vector for expression of p55 gag. This construct is known as pOTSKF33-p55. The Clal-Bglll restriction fragment was isolated from the BH10 clone of the HTLVIIIB isolate of HIV [Shaw et al, Science,
  • Plasmid pDPT287 is a derivative of the chloramphenicol-resistant pDPT270 plasmid of the incFII incompatibility group, which can co-exist with the
  • pDPT287 differs from pDPT270 by two deletions, one removing a small HindiII fragment between the streptomycin-resistance gene and the incFII origin of replication and the other removing a Hindi fragment between the chloramphenicol and
  • Plasmid pDPT287-p55 was constructed by excising the entire P L -p55 transcriptional cassette from
  • pOTSKF33-p55 described in IV above, on a Bgllll-Sall filled-in restriction endonuclease fragment and inserting it between the unique BamHI and HindiII filled-in sites of pDPT287. This deletes the streptomycin-resistance gene of the vector. The orientation of the fragment with respect to the plasmid markers did not influence the expression of the galK-p55 product.
  • bacteria were co-transformed with the p55-expressing vector and each of the PRO constructs separately. Both expression plasmids were induced simultaneously, and the extracts were analyzed by
  • PRO4-BX contained the four-codon insertion that eliminated protease
  • Plasmid PRO4-BS was derived from the PRO4 expression vector by digestion with Bel I, treatment with DNA polymerase (Klenow), and ligation with the 12-mer oligonucleotide 5" CATGTTAACTAG 3", which introduces stop codons in all three reading frames. This treatment resulted in the interruption of the protease coding sequence between the conserved domains.
  • constructs are proteolytically active in both their precursor and mature forms and can carry out the
  • the processed 24-kDa gag protein was purified from induced cells, and its amino-terminal sequence was determined.
  • E. coli cells co-expressing the PRO4 and pDPT287-p55 constructs were disrupted by sonication in
  • the p24 protein eluted in the range of 48-54% (vol/vol) acetonitrile and was approximately 90% pure as judged by Coomassie blue-stained NaDodSO 4 /polyacrylamide gel.
  • a plasmid expressing HIV-1 protease not requiring auto-processing for proteolytic activity was constructed as follows:
  • the PRO5 construct was derived from the PRO4 construct, described in Example 1, by introduction of a translation initiation codon immediately before the first codon of the mature protease (Proline 1) and a translation termination codon immediately after the last codon of the mature protease (Phenylalanine 99). This was done by site-specific mutagenesis using oligonucleotides with the following sequences:
  • the PRO5 insert a 375 bp Ndel-Haelll fragment, has the following nucleotide sequence and corresponding translation for the reading frame of interest:
  • the bacterial expression vector, pSKF-Pr-bglo, which produces a fusion protein consisting of the first 56 amino acids of galK, followed by a cleavage sequence for the HIV protease and ending with the full-length coding sequence for the beta-chain of human hemoglobin was constructed as follows:
  • pSKF-bglo was linearized with Neol and an oligonucleotide with Neol overends

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Abstract

L'invention décrit des procédés pour fabriquer des produits protéiques matures. Une cellule hôte qui a été co-transformée avec deux molécules d'ADN de recombinaison, la première de ces molécules renfermant une séquence de nucléotides de protéine de fusion, cette protéine de fusion encodée par ladite séquence de codage comprenant une première partie, une partie produit protéique et entre les deux une partie site de clivage de protéolytique, et la seconde desdites molécules d'ADN de recombinaison comprenant une séquence de codage pour une protéase ou bien un fragment actif ou un dérivé de celle-ci, susceptible de cliver l'aminoacide encodé par la partie site de clivage protéolytique, est mise en culture, et lors de la mise en culture de la cellule, la protéine de fusion et la protéase sont exprimées. La protéase clive la protéine de fusion au niveau du site de clivage protéolytique pour ainsi produire le produit protéique. Dans des formes d'exécution préférées de l'invention, la séquence de nucléotides de la protéine de fusion et la séquence de nucléotides de la protéase sont incorporées dans deux vecteurs d'expression qui sont ensuite introduits dans la même cellule en vue de l'expression et de la production du produit protéique mature.
PCT/US1989/005009 1988-12-09 1989-11-07 Utilisation d'un site de clivage de protease de vih pour exprimer des proteines matures WO1990006369A1 (fr)

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
WO1991016436A1 (fr) * 1990-04-13 1991-10-31 Schering Corporation Nouveaux dosages de proteases
US5599906A (en) * 1990-04-13 1997-02-04 Schering Corporation Protease assays
US6077694A (en) * 1990-09-21 2000-06-20 The United States Of America As Represented By The Department Of Health And Human Services Method for over-expression and rapid purification of biosynthetic proteins
WO2004074488A1 (fr) * 2003-02-21 2004-09-02 Biotecnol S.A. Utilisation d'enzymes caspases pour la maturation de fusions polypeptidiques de recombinaison par genie genetique
US7169603B2 (en) * 2000-07-14 2007-01-30 Mgi Pharma Biologics, Inc. α-MSH related compounds and methods of use

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CELL, Volume 40, published January 1985, S. WAIN HOBSON et al., "Nucleotide sequence of the AIDS virus, LAV", pp. 9-17, see Figure 1 and Results section entitled "pol". *
DNA, Volume 3, Number 5, published 1984, M. HEUSTERSPREUTE et al., "Expression of galactokinase as a fusion protein in Escherichia coli and Saccharomyces cerevisae", pp. 377-386, see entire article. *
J. BIOL. CHEM., Volume 253, Number 10, published 25 May 1978, T. YOSHIMOTO et al., "Post-proline cleaving enzyme and post-proline dipeptidyl aminopeptidase", pp. 3708-3716, see abstract, Tables I, II, III. *
PROC. NATL. ACAD. SCI. USA, Volume 84, published December 1987, C. DEBOUCK et al., "Human immunodeficiency virus protease expressed in Escherichia coli exhibits autoprocessing and specific maturation of the gag precursor", pp. 8903-8906, see entire article. *
PROC. NATL. ACAD. SCI. USA, Volume 84, published July 1987, V.K. CHAUDHARY et al., "Activity of a recombinant fusion protein between transforming growth factor type alpha and Pseudomonas toxin", pp. 4538-4542. See Results section. *
VIROLOGY, Volume 161, published September 1987, O.C. RICHARDS et al., "Formation of poliovirus RNA polymerase 3D in Escherichia coli by cleavage of fusion proteins expressed from cloned viral cDNA", pp. 348-356, see entire article. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991016436A1 (fr) * 1990-04-13 1991-10-31 Schering Corporation Nouveaux dosages de proteases
US5599906A (en) * 1990-04-13 1997-02-04 Schering Corporation Protease assays
US5721133A (en) * 1990-04-13 1998-02-24 Schering Corporation Protease assays
US5891635A (en) * 1990-04-13 1999-04-06 Schering Corporation Protease assays
US6077694A (en) * 1990-09-21 2000-06-20 The United States Of America As Represented By The Department Of Health And Human Services Method for over-expression and rapid purification of biosynthetic proteins
US7169603B2 (en) * 2000-07-14 2007-01-30 Mgi Pharma Biologics, Inc. α-MSH related compounds and methods of use
WO2004074488A1 (fr) * 2003-02-21 2004-09-02 Biotecnol S.A. Utilisation d'enzymes caspases pour la maturation de fusions polypeptidiques de recombinaison par genie genetique

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