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WO1999067381A1 - TRAITEMENT D'INFECTIONS AUX $i(STAPHYLOCOQUES) - Google Patents

TRAITEMENT D'INFECTIONS AUX $i(STAPHYLOCOQUES) Download PDF

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WO1999067381A1
WO1999067381A1 PCT/US1999/014073 US9914073W WO9967381A1 WO 1999067381 A1 WO1999067381 A1 WO 1999067381A1 US 9914073 W US9914073 W US 9914073W WO 9967381 A1 WO9967381 A1 WO 9967381A1
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gene
lysostaphin
modified
glycosylation sites
eukaryotic
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PCT/US1999/014073
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WO1999067381A9 (fr
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John A. Bramley
Karen I. Plaut
David Kerr
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University Of Vermont And State Agricultural College
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Priority to AU47067/99A priority Critical patent/AU4706799A/en
Publication of WO1999067381A1 publication Critical patent/WO1999067381A1/fr
Publication of WO1999067381A9 publication Critical patent/WO1999067381A9/fr
Priority to US10/087,667 priority patent/US6875903B2/en

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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Bovine staphylococcal mastitis is a frequent problem for the dairy industry, and leads to estimated annual economic losses of $ 184 per cow per year. This corresponds to a U.S. total of $1.7 billion per year for milk producers and milk processors. These losses arise from reduced milk yield, reduced compositional quality, lower product quality and increased veterinary cost.
  • Staphylococcus aureus (S. aureus) is a major pathogen that infects both humans and animals, and that accounts for 15 to 30% of intramammary infection cows. Staphylococcus infections are often characterized by their persistence and their deleterious effects on milk production and quality. Current therapies and preventative treatments for staphylococcal mastitis rely heavily on sterilization techniques, selective culling of animals with chronic recurring mastitis, and the use of ⁇ -lactam antibiotics such as cepharin and penicillin derivatives
  • the present invention provides an improved approach for the treatment of microbial infections in mammals.
  • the invention provides methods and reagents for expressing in mammalian cells microbial proteins that have anti-microbial, particularly anti-staphylococcal, activity.
  • the invention provides both altered genes, in which the naturally-occurring microbial sequences have been engineered to allow expression of active protein in desired mammalian cells or tissues, and methods of introducing such altered genes into desired mammalian cells and/or tissues.
  • an altered gene is modified in such a manner that the protein it encodes is not only produced in mammalian cells, but is secreted from those cells, so that a local concentration of anti-staphylococcal protein is created outside of the cells.
  • such cells either are, or are in the vicinity of, cells that are targeted by infectious microbes S. aureus for attachment and penetration.
  • an altered gene is prepared so that the anti-microbial protein is expressed within cells that are sensitive to intracellular infection.
  • the teachings of the present invention are particularly applicable to treatment of staphylococcal mastitis infections in ruminants, such as cows, goats, and sheep, and most particularly in cows.
  • the altered gene is delivered to mammary tissue in a form and through a mechanism that allows transient transfection of certain cells, preferably localized within the lining of the teat.
  • the altered gene is delivered through the production of a transgenic animal.
  • any of a variety of anti-microbial agents may be employed according to the present invention, but one particularly preferred agent is lysostaphin.
  • the natural lysostaphin gene is altered to contain one or more of a mammalian promoter, transcriptional regulatory sequence, transcriptional termination signal and/or polyA site, splicing sequences, and translation initiation sequences.
  • Preferred altered genes also include sequences that mediate lysostaphin export from the mammalian cells in which the protein is expressed.
  • Particularly preferred altered genes contain sequence modifications that disrupt one or more post-translational processing events that would otherwise occur upon expression of the lysostaphin protein in the mammalian cells.
  • Figure 1 is a schematic representation of the prepro lysostaphin polypeptide.
  • Figure 2 is a representation of modifications of the lysostaphin gene for eukaryotic expression.
  • Figure 3 is an experiment demonstrating lysis of S. aureus by bioacitve lysostaphin produced by COS-7 cells transfected with pCMV-Lys. Conditioned media or cell extracts were lyophilized and resuspended with the original volume (IX), 0.5 volume (2X), or 0.25 volume (4X) of H 2 O. Lysostaphin standards were prepared in media. Samples or standards (15 ul) were then applied to an LB agar plate freshly streaked with S.
  • top row lysostaphin standards at concentrations of 10, 33, 100, and 333 ug/ml
  • middle row blank, IX, 2X, 4X cell extract
  • bottom row blank, IX, 2X, 4X conditioned media.
  • Figure 4 is a Western blot analysis of conditioned media samples treated with or without N-glycosidase-F. Molecular size standards are shown on the left. Samples in lanes 1,2, and 3 were untreated, and contain 20 ul of media from COS-7 cells transfected with: pCMV-hGH control, spiked with 1 ug/ml lysostaphin (lane 1), pCMV-hGH control (lane 2), pCMV-hGH-Lys (lane 3). Samples in lanes 4 and 6 were incubated with deglycosylation buffers, no enzyme, for 16 hr, 37°. Sample in lane 5 was incubated with deglycosylation buffers, and enzyme, for 16 hr, 37°. Lanes contain 20 ul of media from COS-7 cells transfected with; p CMV-hGH (lanes 4), pCMV-hGH-Lys (lanes 5 , 6).
  • Figure 5 is a bacterial plate assay for detection of lysis of S. aureus (M60) by media or cell extracts from transfected COS-7 cells.
  • Media and cell extracts were obtained 48 hr post transfection with (1) pCMV-Lys, (2) pCMV-hGH-Lys, (3) pCMV- hGH-Lys- ⁇ Glyl- ⁇ Gly2, (4) pCMV-hGH genomic as a control.
  • Conditioned media or cell extracts were lyophilized and resuspended with one third the original volume of H 2 O.
  • Top row Lysostaphin standards at concentrations of 11 , 33, 100 or 300 ng/ml in media.
  • Second row Conditioned media
  • Third row cell extracts.
  • Bottom row Lysostaphin standards that were diluted 1 :3 in media, lyophilized, and resuspended with one third the original volume of H 2 O.
  • Figure 6 represents western blot analysis of lysostaphin expressed in transfected COS-7 cells. Proteins were separated on a 12% polyacrylamide-SDS gel, transferred to nitrocellulose membranes and probed with a rabbit anti-lysostaphin polyclonal antibody.
  • Lanes contain 50 ⁇ l cell extract (CE), or media (M), respectively, from cells transfected with: pCMB-hGH as control (lanes 1,2), pCMV-hGH-Lys (lanes 3,4), pCMV-hGH-Lys- ⁇ Gly2 (lanes 5,6), pCMV-hGH with standard lysostaphin protein added to 1 ⁇ g /ml (lanes 7,8), Ad- prepro lysostaphin (lanes 10,11). Lane 9 (*) contained culture media and Ad- preprolysostaphin as used for infection.
  • CE cell extract
  • M media
  • Figure 7 shows tissue fragments, tissue section and cultured cells, exposed to the X-gal reagent for visualization of ⁇ -galatosidase activity (panel A-E).
  • A Teat tissue
  • B adjacent mammary tissue
  • C sections of teat tissue
  • D primary culture of mammary tissue from one goat mammary gland transfected with LacZ-containing adenovirus (AvlLac4) by intrammary infusion. The contralateral gland was infused with vehicle and blue staining was not observed (lower tissue pieces panel A and B).
  • F Green fluorescent protein in COS-7 cells transfected with the GFP gene.
  • Figure 8 depicts lysis of S. aureus by bioactive lysostaphin produced by 293 cells infected with Ad-hGH-Lys- ⁇ GLYl- ⁇ GLY2. Lysostaphin standards were prepared in media. The concentrations were 3 ng/ul, 30 ng/ul and 100 ng/ul. Samples (60 ul) or standards (15 ul) were added to a LB agar plate freshly streaked with S. aureus. Results were evaluated following a 12 hour incubation at 37°.
  • Top row from left to right, lysostaphin standards at concentrations of 3, 100, 30 ng/ul; middle row, from left to right: cell culture media of 293 cells infected by Ad-hGH-Lys- ⁇ GLY1- ⁇ GLY2 isolates #4 and #6, and by 293 cells infected with parent virus Addl 327.
  • Figure 9 shows a Western blot of lysostaphin in milk from transgenic mice containing the BLG-Sec-Lys- ⁇ fl v2 construct.
  • Lane 1 contains 10 ⁇ l of lysostaphin standard (Sigmas 1 ⁇ g ⁇ l) in PBS - 1% BSA.
  • Lanes 2-4 contain milk samples from three different transgenic mice (lO ⁇ l milk diluted 1 :10 in PBS - 1% BSA) Lysostaphin was not detected in non-transgenic mouse milk.
  • Figure 10 shows a bacterial plate assay for lysostaphin bioactivity in mouse milk. Milk samples or lysostaphin standards (15 ⁇ l) were spotted onto a freshly plated lawn of
  • Figure 11 depicts the entire DNA sequence of the lysostaphin gene cloned by
  • FIG. 12 depicts the prepro lysostaphin amino acid sequence encoded by the lysostaphin gene cloned by Recsei et al., supra.
  • Figure 13 depicts the DNA coding sequence of the mature lysostaphin protein of the present invention containing 12 amino acid substitutions as compared to the Recsei et al. (supra) sequence.
  • the modified sequence encodes a 246 amino acid protein in which all but 2 amino acids are identical to the protein encoded by the Recsei et al.
  • Figure 14 depicts the DNA sequence of the ⁇ -lytic protease gene (Li et al., J.
  • FIG. 15 depicts the lysostaphin DNA sequence cloned by Heinrich et al., (Mol.
  • Figure 16 depicts the DNA sequence of the lysostaphin gene cloned by Thumm and Gotz et al., (Molecular Microbiology, 23:1251-1265, 1997). The sequence presented encodes three bacterial genes. Lysostaphin is encoded by nucleotides 725-2018 of the
  • Figure 17 depicts the amino acid sequence of the lysostaphin gene cloned by (Thumm and Gotz et al., supra).
  • SEQ ID NO:l is the sequence of the naturally-occurring lysostaphin gene of
  • SEQ ID NO:2 is the sequence of the naturally-occurring lysostaphin protein. The sequence presented is of the preproprotein ( Figure 12).
  • SEQ ID NO:3 is the sequence of an inventive altered lysostaphin gene ( Figure 13).
  • SEQ ID NO:4 is the ⁇ -lytic protease gene from Achromobacter lyticus ( Figure 14).
  • SEQ ID NO: 5 is a second sequence of a naturally-occurring lysostaphin protein (Heinrich et al., Mol. Gen. Genetic. , 209:563-569, 1987) ( Figure 15).
  • SEQ ID NO: 6 is a third sequence of the naturally-occurring lysostaphin protein
  • altered gene An “altered” gene, as that term is used herein, is identical to a naturally-occurring gene except that the nucleotide sequence of the altered gene has been modified with respect to that of the naturally-occurring gene through the addition, deletion, substitution, or inversion, of one or more nucleotide residues.
  • Preferred altered genes are those in which the coding sequence of a microbial anti-staphylococcal agent is operatively linked with mammalian expression sequences.
  • Particularly preferred altered genes are those in which at least a portion of the microbial sequence (sufficient to encode a protein with anti-staphylococcal activity) is linked to sequences that direct the secretion of the protein from mammalian cells.
  • Such preferred altered genes may also include sequence modifications that remove (or add) sites for post-transcriptional modifications that would otherwise occur in the mammalian cells.
  • a "gene” includes expression signals as well as coding sequence.
  • Gene Generally speaking, a “gene”, as used herein, is a single transcription unit. However, as will be clear from context and is understood in the art, the term can be used in more than one way. The “gene” for a particular protein always includes the sequence that actually encode the protein. A “gene” may also include regulatory sequences such as sites recognized by transcriptional regulators, or responsible for transcriptional termination. A “gene” may also include intronic sequences and/or splicing signals. "Microbial host”: The term “microbial host” is any self-replicating host of microscopic size that encodes within its nucleic acid genome, an anti-microbial agent. As used herein, “microbial host” can also refer to a plants and fungi that encode within their nucleic acid genome an antimicrobial agent useful in the present invention.
  • Naturally-occurring is sometimes used herein to describe microbial genes encoding agents with anti-staphylococcal activity and is intended to refer to the form of the gene (i.e., the gene sequence) that is present in nature, in the microbial host in which the gene is found. Any self-replicating entity that contains nucleic acid and is found in nature can be a "microbial host” for the purposes of this definition. Moreover, although it is not generally so used in common parlance, the term “microbial host”, as used herein, may refer to a plant host.
  • operatively linked is used herein to refer to nucleic acid sequences that are associated with one another in such a way that they are operative with respect to one another.
  • a promoter is operatively linked to a gene coding sequence when it is associated with that sequence in a manner that allows it to direct transcription of that sequence.
  • operative linkage involves covalent attachment via a 3 -5' phosphodiester bond.
  • recombinant refers to a nucleic acid or protein that is produced using the established techniques of recombinant DNA technology (i.e., digestion with restriction endonuclease, ligation, site-specific DNA mutation, polymerase chain reaction, etc.).
  • a recombinant protein is one that is produced from a gene that was made thereof, or from replicative progeny thereof.
  • regulatory sequence is a region of DNA that, when altered or deleted, has an effect on the expression level of the gene with which it is operationally linked. Typically, regulatory sequences are regions of DNA that are recognized (i.e., bound by) protein factors that participate in the regulation of gene expression.
  • the present invention provides altered versions of microbial genes that encode agents with anti-microbial activity, the versions having been modified so that they direct expression of active protein in mammalian tissues or cells.
  • microbial proteins naturally found in any number of microbial hosts, are known to have anti-microbial activity.
  • the gene encoding any such protein could be altered in accordance with the present invention.
  • Preferred genes include those encoding anti- staphylococcal activity, for example, ⁇ -lytic protease, lysostaphin, -lytic protease, lyt-M, atlALE-1, zoo A.
  • Other preferred anti-microbial peptides or proteins whose genes could be utilized include lysozyme, nisin, muramidases, glucoasminidases, and colicins. (see, for example Shockman and Barrett, Proc.
  • bacteriocins which are peptide antibiotics that are produced by bacteria and are effective against even closely related species but do not have significant deleterious effects on the species that produces them or on eukaryotic cells.
  • bacteriocin gene is the lysostaphin gene. ALTERED LYSOSTAPHIN GENES
  • Lysostaphin is naturally produced by Staphylococcus simulans. Lysostaphin kills closely related staphylococcal species, but does not harm other bacterial species or eukaryotic cells. Lysostaphin has endopeptidase activity and kills cells by hydrolyzing the pentapeptide links of staphylococcal cell walls, causing the cells to lyse (Schindler and Schuhardt, Proc. Natl. Acad. Sci. U.S.A., 51 :414-421,1964). If injected directly into the mammary gland of mice or dairy cattle, recombinant lysostaphin is protective against staphylococcal infection (Bramley and Foster, Res. Vet.
  • the minimum inhibitory concentrations of recombinant lysostaphin against S. aureus are less than 100 ng per ml in culture media and less than 2 ⁇ g per ml in milk, (Bramley and Foster, Res. Vet. Sci., 49:120-121, 1990; Oldham and Daley, J. Dairy Sci., 74:4175-4182, 1991).
  • This low concentration requirement makes lysostaphin an attractive candidate for the prevention and treatment of mastitis, because one requirement of the present invention is that the protein be expressed and secreted at sufficient concentrations in vivo to kill S. aureus.
  • preprolysostaphin is 493 amino acids having a signal peptide of 36 amino acids, a propeptide of 211 amino acids and a mature lysostaphin protein of 246 amino acids.
  • mature form refers to a lysostaphin protein which has had the propeptide cleaved off.
  • active forms of lysostaphin are not limited to the mature form; other unprocessed forms of lysostaphin also have activity.
  • preprolysostaphin and prolysostaphin are bioacitve, but mature lysostaphin is 4.5 times more bioactive than prolysostaphin (Thumm and Gotz et al., supra). Variations of lysostaphin that can be modified to be expressed in an active form in mammalian cells fall within the scope of the presently claimed invention.
  • the naturally-occurring lysostaphin gene sequence must be modified to allow for expression of active lysostaphin protein in mammalian cells.
  • expression of bacterial proteins in mammalian cells is often not trivial.
  • the bacterial coding sequence must be operatively linked to a mammalian, or at least a eukaryotic, promoter and a eukaryotic translation initiation sequence.
  • coding sequence Although it is often not required that every nucleotide of coding sequence be preserved, or that the coding sequence initiate and terminate at precisely the same points as it does in its natural host system (fusion proteins and modest deletions are usually tolerated), it is essential that the coding sequence to be employed be operatively linked to expression signals that are effective in the cells into which the altered gene is to be introduced.
  • eukaryotic, and particularly mammalian, expression signals include promoters, transcriptional regulatory sequences (often provided in conjunction with the promoter with which they are naturally associated, or with a promoter with which they have previously been experimentally associated), transcriptional termination signals, splicing signals, translation initiation signals, post-transnational processing signals, and secretory signals (see, for example, Current Protocols 16.0 - 16.21.9).
  • promoters include promoters, transcriptional regulatory sequences (often provided in conjunction with the promoter with which they are naturally associated, or with a promoter with which they have previously been experimentally associated), transcriptional termination signals, splicing signals, translation initiation signals, post-transnational processing signals, and secretory signals (see, for example, Current Protocols 16.0 - 16.21.9).
  • transcriptional regulatory sequences of transcription and translation initiation signals
  • other sequences may be employed as necessary or desirable.
  • Various other modifications may also be made.
  • Promoters that may be employed include constitutive promoters, inducible promoters, universal promoters (i.e., active in substantially all cell types), and/or tissue specific promoters. Those of ordinary skill in the art will appreciate that the precise application of the inventive altered gene will determine which category of promoter is more desirable. For example, if expression is desirably limited to a particular tissue, a tissue-specific promoter is employed; if expression is desirably limited to times when certain environmental conditions are present, an inducible promoter responsive to those environmental conditions is employed. Particular promoters are also selected on the basis of their ability to direct higher or lower levels of transcription.
  • an altered lysostaphin gene is to be expressed in mammary tissue. If the altered gene is to be introduced only into mammary cells, a tissue-specific promoter is not required. Preferred promoters for use in such circumstances include, but are not limited to, Cytamegalo virus, (CMV), Rous Sarcona Virus (RSV) and human elongation factor 1 (EF-1) ⁇ subunit. Particularly preferred is
  • tissue-specific promoter may nonetheless be employed.
  • mammary-specific promoters include, for example, ⁇ -lactoglobulin, ⁇ -lactalbumen, caseins and whey acidic protein. Particularly preferred is the ⁇ -lactoglobulin promoter.
  • the Kozak sequence is well established to be the eukaryotic translation initiation sequence and is the preferred sequence to be introduced into altered genes of the present invention.
  • the low level of activity detected by William et al. (supra) (less than 1 ng/ml) is likely due either to release from lysed cells and a small amount of protein that escapes glycosylation in the in vitro system.
  • Example 1 and Figure 2 we have prepared an altered version of the lysostaphin gene that directs production and secretion of active lysostaphin from mammalian cells.
  • Our first attempt at producing an active, secreted lysostaphin in mammalian cells utilized a construct, pCMV-Lys, in which the coding sequence for mature lysostaphin was operatively linked to the cytomegalovirus promoter and the bovine growth hormone polyadenylation signal.
  • This construct like that described by Williamson et al., was sufficient to produce lysostaphin in mammalian cells, but did not produce active secreted protein (Figure 3).
  • pCMV- hGH-Lys that included a mammalian signal peptide to direct secretion of the lysostaphin protein from the cell.
  • signal peptides including, but not limited to, ⁇ -lactoglobulin, caseins, erytropoietin, and insulin, so long as they were linked in-frame to the lysostaphin coding sequence.
  • We elected to use the human growth hormone signal peptide because expression and secretion of the entire human growth hormone gene had previously been demonstrated in the ruminant mammary gland (Kerr et al., Anim.
  • the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophan and methionine.
  • the polar (hydrophilic), neutral amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • an "altered lysostaphin gene” of the present invention is a lysostaphin gene whose sequence has been modified as compared with that of naturally-occurring lysostaphin (SEQ ID NO:3) in that lysostaphin coding sequence sufficient to encode at least mature lysostaphin has been (i) operatively linked to mammalian expression signals sufficient to direct expression of the gene product in mammalian cells; (ii) operatively linked to a mammalian signal peptide such that the expressed gene product is secreted from the mammalian cells in which it is produced, and, preferably, (iii) modified such that at least one, and preferably both, of the Asn-X- (Ser/Thr) N-linked glycosylation sites is disrupted.
  • the lysostaphin coding sequence that is useful in the production of altered lysostaphin genes according to the present invention is not limited to the mature lysostaphin sequence; the preprolysostaphin and prolysostaphin sequences have also been shown to produce active proteins, although expression of the inmature form of lysostaphin is substantially less than that of the mature form..
  • various changes to the precise lysostaphin amino acid sequence can readily be made without interfering with (and sometimes promoting, as seen in the glycosylation site examples) lysostaphin activity.
  • the sequence may be used in accordance with the present invention.
  • Those of ordinary skill in the art are well familiar with techniques for modifying amino acid sequences (see, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N. Y., incorporated herein by reference), and may employ any known technique, including those described herein, to assay the proteins produced from genes containing such modifications in order to determine whether such genes encode functional proteins as required by the present invention. Altered genes that direct expression and serection of an active lysostaphin protein with one or more sequence differences from naturally-occurring lysostaphin
  • SEQ ID NO:l or from the particular altered lysostaphin described herein (SEQ ID NO:3) are considered to be "functional equivalents" of the altered lysostaphin described herein, and are within the scope of the present invention.
  • lysostaphin gene Additional modifications to the lysostaphin gene that fall within the scope of the present invention include, for example, nucleotide substitutions that more accurately reflect eukaryotic codon usage without altering the amino acid sequence of the encoded protein (see, for example, (Sambrook, J. et al., supra). Such changes are expected to enhance the efficiency of translation and the amount of protein being produced.
  • Another modification involves removal or disruption of a potential polyadenylation signal near the 3' end of the lysostaphin gene.
  • ⁇ -lytic protease gene (SEQ ID NO:4) from Achromobacter lyticus (Li et al., J. BacterioL, 172:6506-6511, 1990).
  • ⁇ -lytic protease exhibits potent bacteriolytic activity on Micrococcus lysodeikticus and S. aureus. It is approximately 25-fold more potent than lysostaphin on heat killed S. aureus, and approximately 40-fold more potent than lysostaphin on viable S. aureus (Li et al., J. Biochem. (Tokyo), 122: 772-778, 1997).
  • An altered ⁇ -lytic protease gene according to the present invention is produced, as was the case with the lysostaphin gene, by operatively linking ⁇ -lytic protease coding sequence with (i) a mammalian promoter; (ii) a mammalian translation initiation sequence; and (iii) a mammalian signal peptide. Additional modifications may also be made.
  • Any of a variety of other genes encoding agents with anti-microbial activity may also be employed in accordance with the present invention. As discussed above, a variety of different microbial anti-staphylococcal agents are known. Any gene encoding such an agent may be modified as described herein to produce an altered gene of the present invention. Useful genes may be isolated from any natural source, including bacteria, fungi, plants, and other microbes.
  • genes are modified to produce altered genes of the present invention through operative linkage with (i) a mammalian promoter; (ii) a mammalian translation initiation sequence; and (iii) a mammalian signal peptide. Additional modifications may also be made.
  • some such genes may have introns, or sequences that are recognized as introns, that are inappropriately spliced in a mammalian system.
  • Such inappropriate splicing events can be identified, for example, by isolating mRN A from a mammalian cell transfected with a version of the gene that has been modified to include the mammalian promoter, translation initiation sequence, and signal peptide. Inappropriate splicing may be corrected by alteration of inappropriate splice sites, or removal of intronic sequences. However, it is often desirable to maintain
  • intron-containing genes are often more efficiently expressed in mammalian systems (see, for example, Wall and Seidel, Theriogenology, 38:337-357).
  • the mammalian signal peptide might not be properly cleaved from the protein produced upon expression of a modified gene containing a mammalian promoter, translation initiation sequence, and signal peptide in a mammalian cell.
  • Inappropriate signal peptide cleavage may be identified by immunopurification of the expressed protein, which is then analyzed by polyacrylamide gel electrophoresis and/or N-terminal sequencing. Problems with signal peptide cleavage can generally be corrected through selection of a different signal peptide, such as one from one of the major milk proteins.
  • modifications may be made to introduce mammalian codons without changing protein sequence, to remove or disrupt any putative glycosylation sites and/or polyadenylation signals, etc.
  • the altered anti-microbial genes of the present invention may be introduced into mammalian cells or tissues in order to treat, or prevent, infection of those tissues.
  • In vitro transfection methods that introduce DNA into mammalian cells in culture are well known in the art, and include calcium phosphate transfection, DEAE-dextran transfection, electroporation, and liposome-mediated transfection.
  • the method of protein expression utilize methods that transfer DNA into living cells in vivo.
  • the genes are delivered by somatic cell engineering, or gene therapy. In such circumstances, the genes are not delivered to the animal's offspring, and are often (unless retroviral delivery systems are employed) only transiently expressed in the cells to which they are delivered.
  • Such systems include, but are not limited to, high-pressure jet injection, lipisome-based delivery systems, and viral delivery systems, including both retroviral and standard viral systems.
  • the mammalian cells and tissue into which altered genes of the present invention are to be produced include any mammalian cells or tissues.
  • Preferred cells are tissues within ruminants such as cows, sheep and goats, but also include human tissues.
  • mammary tissue is one particularly preferred tissue for expression (see below), any tissue that is susceptible to, or that is experience, microbial infection, is a desirable expression site according to the present invention.
  • mammary tissue is a particularly preferred aspect of the present invention.
  • the delivery system is selected, in combination with the gene modifications, to ensure that the altered gene is expressed in mammary tissue.
  • an altered anti-staphylococcal gene is delivered locally to mammary epithelial cells via the teat canal. This route of intramammary infusion administration has the greatest chance of transfecting the epithelial cells lining the teat or teat duct, which are a prime target for attachment and invasion by staphylococcal species and therefore are also an important target for the production of anti-microbial proteins.
  • delivery of plasmid DNA into lactating sheep mammary parenchyma can be achieved by high-pressure jet-injection. This method achieves transfection of cells within the narrow path of the injectate (Kerr et al., Anim. Biotechnol., 7:33-45, 1996). Altered anti staphylococcal genes can be delivered to the epithelial cells of the mammary gland by non- viral approaches (Hyde et al., Nature, 362:250-255, 1993; Oudrhiri et al., Proc. Natl. Acad Sci.
  • Non- viral approaches generally rely on liposome carriers to enhance transfection efficiency. For example, transfection of guinea pig mammary gland with the human growth hormone gene resulted in accumulation of up to 500 ng/ml of the human growth hormone in milk (Hens et al., supra).
  • viral vector approaches are utilized to achieve transient transfection of mammary epithelial cells with inventive altered genes. Viral vector approaches include retro- (Kay et al., Science, 262:117-119, 1993), adeno- (Smith et al, Nat.
  • Retrovirus infection results in integration of the viral nucleic acid code into the host cell DNA, causing permanent transfection of that cell.
  • Retroviruses can only infect dividing cells, but have been shown to be capable of transfecting the caprine mammary gland during a period of hormone-induced mammogenesis (Archer et al., Proc. Natl. Acad. Sci. U.S.A., 91:6804-6844, 1994).
  • an adenovirus vector is used to deliver altered anti-staphylococcal genes to bovine mammary epithelial cells.
  • the adenoviral-based method of gene delivery has several advantages over retroviral-based gene delivery in that transfection efficiency is higher, and it can infect non-dividing cells.
  • New adenoviral vectors have also been developed that limit the host antiviral immune response which is common to adenoviral transfection.
  • a strong cellular immune response can greatly reduce the persistency of andenoviral-mediated gene expression and precludes repeated administration of the same vector (Ilan et al., Proc. Natl. Acad. Sci. U.S.A., 94:2587-2592, 1997; Chen et al, Proc.
  • Example 2 Direct administration of adenovirus containing the ⁇ -galactosidase gene to the teat of a goat resulted in intense blue staining of the entire lining of the teat canal (see Figure 7). Mammary tissues were also infected. This finding is readily generalizable to the inventive altered genes, which may therefore also be delivered to ruminant mammary cells through adenoviral transfection (see Example 3).
  • the altered genes of the present invention may also be introduced into mammalian cells through transfer into mammalian germ line cells and subsequent production of transgenic animals.
  • Established methods for such germ line transfer include, but are not limited to, micro injection of DNA into one-cell embryos (Gordon et al., Proc. Natl. Acad.
  • Germ cell engineering has become wholly routine in the area of transgenic mice (Gordon et al, Biotechnology, 5:1183-1187, 1987), and has also been broadly applied to pigs (Wall et al, Proc. Natl. Acad. Sci. U.S.A, 88:1696-1700, 1991), sheep (Wright et al., Biotechnology, 9:830-834, 1991), goats (Ebert et al, Biotechnology, 9: 835-838, 1991), and cattle (Krimpenfort et al, Biotechnology, 9:844-847, 1991).
  • transgenic mice that produced human tissue plasminogen activator in transgenic mouse milk. Any of the techniques described in these references, or otherwise known in the art, may be employed to create transgenic animals in which an altered gene of the present invention has been stably introduced into their genome. Such transgenic animals are useful not only as staphylococcus-resistant creatures, but as bioreactors for the production of anti-staphylococcal agents for use in the treatment of others.
  • Lysozyme is a muramidase to which Gram negative and some Gram positive microorganisms such as S. aureus show varying degrees of resistance (for reviews, see Reiter et al., "Protective Proteins in Milk- Biological Significance and Exploitation ", International Dairy Federation Bulletin #191. IDF, Square Vergote 41, 1040 - Brussels, Belgium; Magga and Murray, J. Dairy Sci., 78:2645-2652, 1995).
  • lactofemn which acts as an antimicrobial through its iron-chelating activity, (Reiter et al, supra), does protect the non-lactating mammary gland from infection by E. coli, although this inhibition is lost at the time of calving (Bramley, J. Dairy Res., 43:205-211, 1976).
  • defensins produced in neutrophils, macrophages and epithelial cells lining mucosal surfaces (Kagan et al., Toxicology, 87:131-149, 1994), also have antibacterial action resulting from their ability to form pores in susceptible cellular membranes.
  • defensin bovine tracheal antimicrobial peptide, (TAP)
  • TAP bovine tracheal antimicrobial peptide
  • All four lysostaphin constructs were generated by a PCR-based technique in which the 5' primer included a 5' Not I restriction site and the 3' primer included a 3' Apa I site.
  • the primers were positioned such that only the coding region and the TGA stop codon of the mature portion of the lysostaphin gene were amplified.
  • pCMLEM pCMLEM
  • the expression plasmid pCMV-Lys was constructed by inserting a short linker sequence 5' to the mature lysostaphin sequence, between the Bam HI and Not I sites of the pcDNA3 polylinker.
  • the short sequence was prepared from two custom 13 base oligonucleotide (Gibco/BRL), and resulted in the addition of a Kozak sequence and a start codon (ATG) to the lysostaphin gene.
  • the expression plasmid pCMV-hGH-Lys was constructed by inserting the human growth hormone (hGH) intron-containing signal peptide coding region, 5' to the mature lysostaphin sequence.
  • This eukaryotic signal peptide was chosen to enhance the secretion of lysostaphin from the cells.
  • We have previously had satisfactory experience with the expression of the entire hGH gene in the ruminant mammary gland Karl et al, Anim. BiotechnL, 7:33-45, 1996), and it has been used by others to direct the secretion of engineered proteins (Pecceu et al., Gene, 97:253-258, 1991).
  • the coding region of the hGH signal peptide included the 5' untranslated region and the first intron of the hGH gene.
  • the intronic sequence was included as there is good evidence that introns increase expression of foreign proteins (Wall and Seidel, Jr., Theriogenology, 38, 337-357, 1992).
  • the modified hGH signal peptide was obtained from a collaborator (Dr. K. Wells, GEML-ARS-USDA, Beltsville, MD).
  • the resulting hGH-lysostaphin sequence codes for the amino acids of the entire hGH signal peptide immediately followed by the entire mature form of lysostaphin. The sequence of this construct was confirmed by DNA sequencing.
  • the expression plasmids pCMV-hGH-Lys- ⁇ Gly2 and pCMV-hGH-Lys- ⁇ Glyl- ⁇ Gly2 were subsequently prepared.
  • a PCR strategy was used to remove glycosylation sites from the mature lysostaphin gene and generate pCMV-hGH-Lys- ⁇ Gly2 and pCMV- hGH-Lys- ⁇ Glyl- ⁇ Gly2.
  • pCMV-hGH-Lys- ⁇ Gly2 removes one of two potential N-linked glycosylation sites within mature lysostaphin.
  • pCMV-hGH-Lys- ⁇ Glyl- ⁇ Gly2 was designed to encode a lysostaphin protein in which both N-linked glycosylation sites (Asn-X-Ser/Thr) have been removed by mutation of the site's Asn codons to Gin codons ( Figure 2).
  • Bacterial proteins are not normally glycosylated, but when expressed in a eukaryotic system, any Asn-Xxx-Ser/Thr sequence of amino acids in a protein has the potential for N-linked glycosylation.
  • ⁇ Gly2 and pCMV-hGH-Lys- ⁇ Glyl- ⁇ Gly2 was constructed in a similar fashion to pCMV-hGH-Lys.
  • the 3' primer for generating the lysostaphin amplicon contained nucleic acid substitutions that resulted in a change from AAT to CAG at the codon for amino acid number 232 of the mature lysostaphin protein. This causes an asparagine to glutamine change in the encoded protein, and thus destruction of the potential glycosylation site.
  • Asn to Gin based on the similar structure and characteristics of their side groups.
  • the plasmid pCMV-hGH-Lys- ⁇ Glyl- ⁇ Gly2 was similarly constructed using a synthetic 5' primer.
  • the Asn to Gin strategy was recently reported as being successful in preventing the glycosylation of a bacterial enzyme that was engineered to be expressed on the cell surface of eukaryotic cells
  • Lysostaphin expression from the four new constructs was evaluated following their transfection into COS-7 cells.
  • the cells were transfected in six-well culture plates, with a CaPO 4 precipitation technique (Sambrook et al., Milecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., 1989, Incorporated herein by reference). Following exposure to the plasmid precipitate, cells were washed and then incubated with 1 ml DMEM containing 10% FBS for 48 hr Media was then collected, cleared by centrifugation and stored (-20°). Cell extracts were obtained by freeze/thaw disruption of the cell monolayer with 0.5 ml phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • Transfection efficiency was monitored visually by co-transfection with a green fluorescent protein expression plasmid.
  • the plasmid, pCMV-GFP was constructed by inserting the GFP encoding fragment from pEGFP-NI (Clontech) into pcDNA3. Plasmid DNA used in transfections contained a 9:1 mixture of the test plasmid and pCMV-GFP. Consistently high (>50%) transfection efficiencies were obtained ( Figure 7F).
  • Lysostaphin bioactivity was not detected using the S. aureus plate assay, in either media or cell extracts from COS-7 cells transfected with pCMV-hGH-Lys.
  • substantial lysostaphin immunoreactivity was observed by western blot assay of media but not extracts of cells transfected with p pCMV-hGH-Lys ( Figure 6, lane 4).
  • the band migrated with an apparent molecular weight of -33 Kd, somewhat larger than the lysostaphin standard that migrated at -28 Kd.
  • lysostaphin accumulates within the cells such that cell extract, but not media cause bacterial lysis. This media likely does contain some lysostaphin resulting from cell lysis, but the concentration is below the detection limits of our assay.
  • bioactive lysostaphin is detected only in media, not in cell extract. Presumably the cell extract contains an amount of bioactive lysostaphin that is below detection. No bioactivity is observed from cells transfected with the construct containing the hGH signal peptide and the unmodified lysostaphin gene.
  • the plasmid AvlLacZ4 (Genetic Therapy Inc; Bethesda, MD), is a replication deficient, recombinant, human type 5 adenovirus that contains the gene for nuclear targeted ⁇ -galactosidase (LacZ) (Smith et al., supra). The E3 region of this adenovirus has been deleted and the ⁇ -galactosidase gene replaces the El a region rendering the virus replication incompetent.
  • Viral stocks were prepared using the 293 packaging cell line (ATCC #CRL-1573). This cell line is a stable transfectant that produces the Ad 5 Ela transcription factor and thus complements the Ela deletion in the recombinant virus.
  • Goats were exposed to AvlLacZ4 to evaluate the ability of the human adenovirus to infect the ruminant mammary gland in vivo.
  • One teat of each goat was infused with 1 ml of a solution (10 mM
  • Evaluation of the infections include monitoring animal health (temperature, respiration, rumination, and post-mortem evaluation) and determining SCC and the presence of bacterial infection.
  • Transfection is evaluated by staining teat and mammary tissues with X-gal reagent and then evaluating them grossly and microscopically. Histopathology is also evaluated. Infusions of 1.9 x 10 10 and 0.6 x 10 10 pfu/ml are administered 72 hr and 24 hr prior to euthanasia, respectively. A minor inflammatory response may develop as evidenced by fluid accumulation in the gland and an elevation in somatic cell count. Rectal temperatures are monitored. Secretions are exmined for bacterial contamination. This characterization allows determination of the lower dose of adenovirus that will ameliorate these symptoms, and yet still provide adequate transfection.
  • Adenoviral-based transfection of the goat mammary gland with an engineered lysostaphin construct Adenoviral-based transfection of the goat mammary gland in vivo has been undertaken as with the AvlLacZ4 infections.
  • Each of two multiparous non-lactating goats were infected in one gland by intramammary teat infusion with the lysostaphin- containing adenovirus.
  • the contralateral gland was infused with the LacZ-containing adenovirus.
  • the goats were euthanized 48 hr post-infusion. Mammary secretions were collected prior to infusion, and at 24 hr and 48 hr post-infusion.
  • Secretions and tissues from the glands were processed as previously described with additional measurements for lysostaphin production and activity as follows.
  • Secretions from the glands, and extracts prepared from tissue fragments were assayed for immunoreactive lysostaphin using western blot and an ELISA that have been developed.
  • sample infranatants Prior to assay, sample infranatants were prepared by two sequential centrifugation steps (15 min, 12,000g, 4°), in which the fluid between the fat layer and pelletable protein and debris was harvested. All secretions were normalized based upon total protein content, determined by a modification of the Lowry method (Nerurkar et al, "Quantification of selected intracellular and secreted hydrolyses of macro phages.
  • Lysostaphin production is also evaluated in tissue sections processed for immunohistochemistry using our rabbit polyclonal antibody to lysostaphin.
  • Immunohistochemical techniques are currently available. Briefly, formalin fixed tissue is embedded in paraffin, and sectioned (6 ⁇ m) by the UVM histology core facility. Slides are then deparafrinized and rehydrated. Endogenous peroxidase are blocked by a 10 min incubation with 0.3% hydrogen peroxide in methanol. Non-specific protein binding is blocked with a 30 min incubation in 10% normal goat serum in 1% BSA-PBS. Sections are be incubated for 60 min with lO ⁇ g/ml of our rabbit polyclonal antibody generated against lysostaphin.
  • Bound antibody is detected with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) subsequently coupled to streptavidin-peroxidase. Then the chromogen, amino ethyl carbazole (AEC) and the substrate (0.6%) peroxide) are added to the sections allowing the development of a red color (Zymed Laboratories, San Diego, Calif.
  • Negative controls are incubated with the primary antibody in the presence of a 100 fold excess of a lysostaphin.
  • Example 3 Adenovirus mediated expression of lysostaphin
  • GTI supplied start up quantities of an E3 region deletion mutant (AD5-rf/327) of the AD5 adenovirus, and the shuttle plasmid pAvS6(Smith et.al, supra) that was used to construct the recombinant adenoviruses carrying the lysostaphin gene.
  • the Not I-Kpn I fragment of the shuttle plasmid contains the inverted terminal repeat and encapsidation signal from the left end of AD5-dB27, the RSV promoter, a multi cloning region for insertion of the gene of interest, the SV40 Poly (A+) signal, and Ad5 sequences from nucleotide 3328 to 6246 that serve as a homologous recombination region.
  • pAvS6-preprolys Two shuttle plasmids were constructed.
  • pAvS6-preprolys was constructed by inserting the 1.5 Kb modified lysostaphin gene from pCMLEM
  • This modified lysostaphin gene contained a Kozak region linked to the preprolysostaphin gene.
  • the other called pAvS6-hGH-Lys- ⁇ GLYl- ⁇ GLY2 contained the human growth hormone signal peptide linked to the modified lysostaphin construct which was obtained from pCMV- hGH-Lys- ⁇ GLYl- ⁇ GLY2.
  • the shuttle plasmids, pAvS6-preprolys, and pAvS6-hGH-Lys- ⁇ GLYl- ⁇ GLY2 were independently used to generate two recombinant adenoviruses, Ad-preprolys and Ad- hGH-Lys- ⁇ GLYl- ⁇ GLY2, respectively.
  • the recombinant viruses were constructed by co-transfection of the linearized shuttle plasmids with the Cla I fragment of Ad5-dl327 into 293 cells. Resulting plaques were purified. Insertion of the lysostaphin genes into the viral genomes was confirmed by polymerase chain reaction.
  • Lys-AGLYl- AGLY2 Lysis of S. aureus (M60) by bioactive lysostaphin produced by 293 cells infected with Ad- hGH-Lys- ⁇ GLYl- ⁇ GLY2 was evaluated by plate assay. Samples (60 ul) or standards (15 ul) were added to a LB agar plate freshly streaked with S. aureus. Results were evaluated following a 12 hour incubation at 37°. Lysostaphin standards were prepared in media. The concentrations were 3 ng/ul, 30 ng/ul and 100 ng/ul. Inhibition of S. aureus growth was observed by the standard preparations and by culture media obtained from 293 cell cultures that had been infected with Ad- hGH- Lys- ⁇ GLYl- ⁇ GLY2. These results are illustrated in Figure 1.
  • Example 4 Production and evaluation of transgenic mice incorporating the lysostaphin gene under control of a mammary specific promoter.
  • This model allows assessment of the functionality of the transgene when incorporated into the genome of an animal, determination of toxicity of the transgenic protein to the lactating mammary gland, and assessment of the effects of the transgene on milk production. The antibacterial properties of milk from these animals can also be measured.
  • a variety of mammary gland and lactation specific promoter regions could be used to direct expression of the lysostaphin gene to the lactating mammary gland. These include, but are not limited to, the regulatory sequences of the casein genes, whey acidic protein, and - lactoglobulin. We chose to use the - lactoglobulin regulatory sequence.
  • the 4.2 Kb 5 '-flanking (promoter) region and 2.1 Kb of the 3-flanking region of the ovine -lactoglobulin (BLG) gene (pBJ41) were obtained from Dr. A.J. Clark (Roslin Institute, UK). These components have been used to direct the production of mg/ml concentrations of foreign proteins into the milk of mice (Archibald et al., Proc. Natl. Acad. Sci. USA 87,5178-5182, 1990) and sheep (Wright et al. ,
  • mice Five lines of mice have now been established. These mice appear normal, are fertile, and are able to raise offspring.
  • milk has been obtained from FI mice representing three of the lines. The milk was collected on day 10 of lactation (Maga et al., J. Dairy Sci. 78: 2645-2652, 1995). The milk samples were immediately frozen (-80) and then shipped to our laboratory on dry ice. Milk was analyzed for lysostaphin immunoreactivity as described for cell culture experiments. Prior to analysis milk samples were diluted (1: 10) in PBS containing 0.5% BSA, then defatted by centrifugation (15 min, 4, 10,000g).
  • Transgenic Ruminants The present invention provides transgenic dairy cows containing a modified lysostaphin gene, although the cost and duration of such an endeavor necessitates preliminary experiments using the much less expensive, and more rapid, transgenic mouse model.
  • mammary-specific promoter may be employed.

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Abstract

La présente invention a trait à une approche améliorée du traitement d'infections microbiennes chez des mammifères. Spécifiquement, l'invention concerne des procédés et des réactifs servant à exprimer des protéines de cellules de mammifère qui possèdent une activité antimicrobienne. L'invention concerne à la fois des gènes qui ont été modifiés pour permettre l'expression, et de préférence la sécrétion, de protéine active dans des cellules ou dans des tissus de mammifère voulu ; et des procédés d'introduction de ces gènes modifiés dans des cellules et/ou des tissus de mammifère voulu. Plus spécifiquement, des gènes codant pour des protéines antistaphylococciques sont apportés à des cellules et/ou des tissus de mammifère par des procédés d'apport de gène, comprenant une thérapie génique et la production d'animaux transgéniques, en vue de traiter une mammite chez des animaux ruminants.
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EP1465990A4 (fr) * 2001-12-21 2006-06-28 Biosynexus Inc Molecule de lysostaphine tronquee a activite staphylolytique amelioree
CN100388949C (zh) * 2005-08-09 2008-05-21 上海高科联合生物技术研发有限公司 一种用于防治牛子宫内膜炎的溶葡萄球菌酶冻干粉剂

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US5011772A (en) * 1988-02-05 1991-04-30 Public Health Research Institute Of The City Of N.Y. High yield protein production system
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WO1987006264A1 (fr) * 1986-04-16 1987-10-22 Public Health Research Institute Of The City Of Ne Expression du gene clone de lysostaphine
US5011772A (en) * 1988-02-05 1991-04-30 Public Health Research Institute Of The City Of N.Y. High yield protein production system
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1465990A4 (fr) * 2001-12-21 2006-06-28 Biosynexus Inc Molecule de lysostaphine tronquee a activite staphylolytique amelioree
AU2002367820B2 (en) * 2001-12-21 2007-11-01 Biosynexus Incorporated Truncated lysostaphin molecule with enhanced staphylolytic activity
CN100388949C (zh) * 2005-08-09 2008-05-21 上海高科联合生物技术研发有限公司 一种用于防治牛子宫内膜炎的溶葡萄球菌酶冻干粉剂

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