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WO1999005289A1 - Analogues de lysostaphine recombinants - Google Patents

Analogues de lysostaphine recombinants Download PDF

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Publication number
WO1999005289A1
WO1999005289A1 PCT/CA1998/000679 CA9800679W WO9905289A1 WO 1999005289 A1 WO1999005289 A1 WO 1999005289A1 CA 9800679 W CA9800679 W CA 9800679W WO 9905289 A1 WO9905289 A1 WO 9905289A1
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Prior art keywords
lysostaphin
gly
analog
recombinant
genetic construct
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PCT/CA1998/000679
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English (en)
Inventor
Marc Gagne
Pierre Chapdelaine
Jacinthe Therrien
Dominic Gagne
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9071-7125 Québec Inc.
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Application filed by 9071-7125 Québec Inc. filed Critical 9071-7125 Québec Inc.
Priority to CA002305624A priority Critical patent/CA2305624A1/fr
Priority to NZ502451A priority patent/NZ502451A/en
Priority to EP98933404A priority patent/EP0998570A1/fr
Priority to AU83285/98A priority patent/AU734683B2/en
Publication of WO1999005289A1 publication Critical patent/WO1999005289A1/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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • 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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Definitions

  • the invention relates to lysostaphin analogs having a molecular structure preventing post- translational modifications while retaining lysostaphin-like biological activity.
  • Such recombinant lysostaphin analogs have at least one modified site, or a rearrangement of at least one site at the level of the amino acid sequence of the mature lysostaphin protein.
  • the invention also relates to DNA sequences encoding the lysostaphin analogs, and recombinant plasmids and host cells for the expression of recombinant lysostaphin analog.
  • Lysostaphin is a bacteriolytic enzyme, a bacteriocin, which is naturally produced only by the bacterial strain Staphylococcus simulans biovar staphylolyticus (NRRL B-2628), and that lysates specifically staphylococcal cells.
  • the lysostaphin endopeptidase is zinc-metalloenzyme that lyses staphylococci by hydrolyzing glycine bonds in the polyglycine cross bridges between glycopeptide chains in the cell wall peptidoglycan of these organisms (Health et al., (1987) FEMS Microbiol. letters, 44: 129-133). Lysostaphin endopeptidase can be used as well as food additive as for human and animal therapeutic application.
  • lysostaphin has been found very effective to treat Staphylococcus aureus mastitis in cows after intramammary injection of this protein (Oldham et al., (1991) J. Dairy Sci. 74:4175-4182) .
  • Lysostaphin accumulates during the stationary phase of S. simulans culture grown under aerobic conditions coordinate hexosaminidase and thiolproteinase (Donham et al . , J. Gen. Microbiol., (1988) 134:2615-2621).
  • the gene for lysostaphin- endopeptidase synthesis and represents 1.5 kbp (Recsei et al., Proc. Nat. Acad.
  • lysostaphin can be produced by fermentation techniques wherein S. simulans is grown in liquid culture.
  • Recombinant DNA techniques where by genes for a variety of proteins can be cloned by insertion into a cloning and expression vector, which can then be introduced into microorganisms for production of the same proteins under recombinant form.
  • Microorganisms such as E.
  • coli Bacillus spp and Streptomyces have been used to produced large amount of recombinant lysostaphin.
  • cloning techniques there have been no reports with evident data relating to such cloning techniques being used to insert the genes encoding lysostaphin into a cloning vector to construct novel vectors which can transfect eukacyotic cells to allow the production of lysostaphin either in vi tro or in vivo .
  • These new vectors can provide new alternative to bacterial production systems of lysostaphin, or can be introduced in vivo into an organ, like mammary glands of commercial milk species, and eliminates or prevents infections caused by staphylococci by local production of the bacteriocin.
  • the biological activity of a protein is dependent upon its structure.
  • the primary structure of a protein i.e. its amino acid sequence
  • secondary e.g. ⁇ -helix or ⁇ -sheet
  • glycosylation modifications, referred to as glycosylation, can dramatically affect the physical properties of the proteins and can also be important in protein stability, secretion, and subcellular localization.
  • Other examples of post-translational modifications of proteins can be included in the groups of phosphorylation, acetylation, methylation, or sialylation.
  • Proper glycosylation can be essential for biological activity.
  • some genes from eukacyotic organisms when expressed in bacteria e.g. E. coli ) which lack cellular processes for glycosylating proteins, yield proteins that are recovered with little or no activity by virtue or their lack of glycosylation.
  • prokaryotic proteins like lysostaphin, which are not naturally modified following their translation into bacteria, even so can be altered by post-translational modifications and become biologically inactive because of these undesirable biochemical changing.
  • glycosylation occurs at specific locations along the polypeptide backbone and is usually of two types: 0-linked oligosaccharides are attached to serine or threonine residues while N-linked oligosaccharides are attached to asparagine residues when there are part of the sequence Asn-X-Ser/Thr, where X can be an amino acid except proline.
  • the lysostaphin which contains glycosylation sites in its amino acid sequence could be rendered biologically inactive if post-transtionnally glycosylated in eukaryotic cells in vi tro or in vivo .
  • European Patent No. 299,978 in the name of Applied Microbiology Inc. there is disclosed the expression of the cloned lysostaphin gene and homologues, which exhibit almost any biological activity.
  • lysostaphin gene in an expression vector could allow synthesis of an analog of lysostaphin and prevent post-translational modifications without altering the activity of the enzyme.
  • One aim of the present invention is to provide recombinant lysostaphin analog having a molecular structure preventing post-translational modifications while retaining lysostaphin-like biological activity.
  • Another aim of the present invention is to provide for genetic constructs carrying a modified lysostaphin gene, wherein the gene encodes for a lysostaphin molecule also modified in such a manner that the bacteriolytic activity is restored when produced by eukaryotic cells.
  • the subject invention relates to analogs of lysostaphin comprising an amino acid sequence of the mature lysostaphin which includes at least one modified site.
  • recombinant plasmids which contain DNA encoding the analogs of lysostaphin from Staphylococcus simulans (NRRL-B2628) and which in transformed prokaryotic cells and transfected eukaryotic cells will express a gene encoding lysostaphin analogs.
  • expression of the DNA encoding the analogs of lysostaphin in in vi tro and in vivo systems are provided.
  • the present invention provides a solution to the problem of non-desired post-translational modifications of recombinant lysostaphin produced by eukaryotic cells, which problem has just been recognized for the first time.
  • the present invention provides a solution to restore the biological activity of recombinant lysostaphin produced by eukaryotic cells .
  • a recombinant lysostaphin analog having substantially an amino acid sequence of mature lysostaphin which includes at least one modified site for inhibiting post-translational modification after production by eukaryotic cells, wherein the modified site is at least one added, changed, substituted, or deleted amino acid residue.
  • the post-translational modification is glycolysation, methylation, disulfide bond formation, acetylation, phosphorylation, or sialylation.
  • the modified site of the recombinant lysostaphin analog is for improving the lysostaphin-like biological activity after production by eukaryotic cells.
  • Such a modified site in accordance with the present invention may be one of the following:
  • the recombinant lysostaphin analog of the present invention include, without limitation, the following : Gly 123 -lysostaphin; Glu x2 -lysostaphin .
  • a DNA sequence encoding a recombinant lysostaphin analog having substantially an nucleic acid sequence encoding mature lysostaphin which includes at least one modified site, and wherein the modified site is a nucleic acid addition, changing, substitution, or deletion.
  • the DNA sequence of the present invention include, without limitation, the nucleic acid sequences encoding the recombinant lysostaphin analog of the present invention.
  • the DNA sequence encoding a recombinant lysostaphin analog having substantially an nucleic acid sequence encoding mature lysostaphin may include a rearrangement of at least one site for inhibiting post- translational modifications.
  • a therapeutic composition comprising a therapeutically effective amount of one recombinant lysostaphin analog or of a mixture of more than one analog of the present invention in association with a pharmaceutically acceptable carrier.
  • a method of producing a recombinant lysostaphin analog of the present invention which comprises the steps of: a) transfecting an eukaryotic cells with an inducible expression vector comprising a coding DNA sequence of the present invention and a signal peptide; b) inducing the expression of the coding DNA sequence to produce the recombinant lysostaphin analog .
  • the eukaryotic cells may be mammalian cells.
  • the method may be an in vi tro or an in vivo method.
  • an in vivo method of producing a recombinant lysostaphin analog of the present invention in the milk of a ruminant mammal comprising the steps of: a) preparing a genetic construct including a DNA sequence encoding the recombinant lysostaphin analog of the present invention and a signal peptide in a liquid carrier to form a liquid complex, b) infusing the liquid complex into a cistern, ductal tree, and/or alveoli of a mammary gland of the ruminant mammal and allowing transfection of the mammary gland to permit expression and secretion of the lysostaphin analog into the milk of the mammal; c) collecting milk from the mammal; and d) purifying the lysostaphin analog from the collected milk.
  • the mammal may be a bovine, a sheep, a goat, or a porcine.
  • the recombinant lysostaphin analog produced in vivo is enzymatically active on staphylococci .
  • the genetic construct is treated to enhance its ability to cross the membrane of an epithelial cell.
  • Such a treatment include, without limitation, the following:
  • polycationic compound • forming a complex between the genetic construct and a polycationic compound, wherein the polycationic compound may be selected from the group consisting of poly-lysine and poly- ornitine;
  • the lipid may be cationic
  • Fig. 1 illustrates a schematic representation of the pCMV-lyso-1 plasmid
  • Fig. 2 illustrates examples of amino acid sequence of lysostaphin analogs in accordance with the present invention
  • Fig. 3 illustrates denaturing polyacrylamide gel of the native and modified lysostaphin produced in an in vi tro cell free transcription and translation system
  • Fig. 4 illustrates polyacrylamide gel of endoglucanase-treated native recombinant lysostaphin produced by transfected COS-7 cells in in vi tro cultured cells;
  • Fig. 5 illustrates Western blot analysis of analog and native mature lysostaphin
  • Fig. 6 illustrates translation of the mature lysostaphin construct and of the mutated lysostaphin constructs ;
  • Fig. 7 illustrates activity of analog (clone 5) and native lysostaphin on S. aureus saturated polyacrylamide gel
  • Fig. 8 illustrates Agarose gel showing the production of specific lysostaphin analog mRNA into transfected mouse mammary glands
  • Fig. 9 illustrates Staphylococcus aureus saturated polyacrylamide gel showing the activity of the recombinant lysostaphin analog produced in vivo into transfected mouse mammary glands.
  • the invention features, in one aspect, a method of producing, in a ruminant mammal, milk containing a recombinant lysostaphin analog.
  • the preferred method involves: a) providing a genetic construct including DNA encoding lysostaphin analogs and a signal peptide, b) mixing the construct with a delivery system to form a complex, c) infusing the complex into a culture well containing eukaryotic cells, or into the mammary gland of the mammal, d) raising the culturing the eukaryotic cells so that the lysostaphin analogs encoded by the construct is expressed and secreted into the culture medium, e) raising the mammal so that the lysostaphin analogs encoded by the constructs are expressed and secreted into the milk produced by the secretory cells of the udder (e.g. ductal tree and the mammary alveolar cells f) obtaining the in vi tro culture medium or the milk from the mammal.
  • the invention also features a composition which includes milk produced according to this method.
  • the mammal is bovine, a sheep, a goat, or a pig.
  • a DNA delivery system complex can be infused into the mammal at any age of sexual maturity.
  • the subject invention provides mature lysostaphin analogs and the modified DNA sequence encoding for these lysostaphin analogs.
  • the present invention provides for recombinant plasmids which have been created by insertion of the 741 base pairs (bp) mutated DNA fragment encoding for the mature form of the lysostaphin analogs.
  • the plasmids are cloning vectors that replicates in various host microorganisms, such as E. coli , and that allow transcription and translation of an active lysostaphin analogs both by in vi tro and in vivo transfected eukaryotic cells.
  • the analogs of modified gene derived lysostaphin are different than the native mature lysostaphin produced by Staphylococcus simulans or than - li ⁇
  • the invention relates to the gene encoding a lysostaphin analogs having a specific number (i.e. a fixed number greater than 0) of mutated (changed) nucleic acid residues per codon, for which correspond a lysostaphin peptide analogs having the same number than changed codon of changed amino acid residues per lysostaphin molecule.
  • a lysostaphin analogs having a specific number (i.e. a fixed number greater than 0) of mutated (changed) nucleic acid residues per codon, for which correspond a lysostaphin peptide analogs having the same number than changed codon of changed amino acid residues per lysostaphin molecule.
  • lysostaphin analogs The production in vivo or in vi tro of lysostaphin analogs by the corresponding mutated gene in transfected eukaryotic cells restore (give back) the enzymatic activity to the lysostaphin analogs.
  • the present recombinant plasmids expressed lysostaphin analogs in high levels in cloned transfected mammalian cells harboring the plasmids. Lysostaphin analogs are produced and secreted by transfected cells and accumulates in vi tro in large quantities in the medium in which the transfected cells are cultured.
  • the present invention provides for genetic constructs, which includes any ubiquitous or inducible promoters active in eukaryotic cells, a signal peptide working in eukaryotic cells, most particularly mammalian cells, and the modified lysostaphin gene.
  • signal peptide is meant a polypeptide which facilitates secretion of the protein to which it is linked.
  • the signal peptide can be naturally occurring in the lysostaphin DNA sequence.
  • the genetic construct can be engineered so that a signal peptide is bonded to the lysostaphin analogs .
  • the invention provides also a convenient and efficient method for directly transferring the modified lysostaphin gene into the mammary gland of a ruminant mammal to produce a lysostaphin analog in the milk of the mammal .
  • the transferred DNA is protected against degradation and the efficiency of gene transfer is increased by complexing the DNA with a DNA delivery system.
  • delivery system cationic, neutral, negatively-charged, polycationic, pH-sensitive lipids, polyion, amphiphilic compounds, and polycationic amino polymer, which can complexes with the genetic constructs and allows and enhance their ability to cross the membrane of a secretory cells (e.g. mammary gland epithelial cells) in vivo and in vi tro . These treatments can improve uptake and nuclear localization of the genetic construct.
  • a secretory cells e.g. mammary gland epithelial cells
  • infusion is meant the introduction of the genetic constructs free or complexed to a delivery system into the cistern or duct of mammary gland through the skin or the streak canal.
  • ductal tree is meant the branched network of tubular structures which conduct milk in a mammary gland.
  • wash canal is meant the papillary duct at the lower end of the teat which leads to the ductal tree
  • secretory cells is meant the epithelial cells of the ducts and alveoli of the mammary gland, or any other cell of the mammary gland able to secrete a recombinant lysostaphin analog into the milk
  • modified gene is meant a deletion, inversion, or base substitutions of at least one nucleic acid of the gene, and mutation of at least one amino acid residue of the lysostaphin.
  • promoter is meant an expression control region and to substantial portion of an element located in the 5 ' sequence naturally upstream from the lysostaphin analog encoding region.
  • the term 5 1 sequence naturally upstream of the lysostaphin analogs encoding region is used to refer to the 5 ' sequence which is upstream of the lysostaphin analogs encoding region in its natural position within the genetic constructs such as its natural position within the genome.
  • useful promoters include the human cytomegalovirus (CMV) immediate early promoter, the Simian Virus 40 (SV40), the Rous Sarcoma Virus (SRV), the adenovirus major late promoter.
  • CMV human cytomegalovirus
  • SV40 Simian Virus 40
  • SRV Rous Sarcoma Virus
  • Other useful promoters include those which naturally drive the expression of mammary-specific genes.
  • the ⁇ si-casein promoters, ⁇ s2 ⁇ casein promoters, ⁇ -casein promoters, ⁇ -casein promoters, ⁇ -lactoglobulin promoters, whey acidic protein promoters, and ⁇ - lactalbumin promoters can be used.
  • the promoter can be operably linked to one or more enhancer elements such that the enhancer elements(s) increases transcription of the gene encoding the lysostaphin analogs.
  • Useful enhancer elements include, without limitation, enhancer elements from CMV, SV40, and the RSV long terminal repeat. Expression of the lysostaphin analog genes can be constitutive or, if desired, inducible by an external stimulus.
  • inducible promoters with steroid hormone-responsive elements examples include the mouse mammary tumor virus long terminal repeat, and heat shock promoters (e.g. hsp 70). Methods for inducing these promoters are described in the literature.
  • corticosteroids e.g. dexamethasone
  • the addition of zinc or cadmium to the ruminant's feed or water will drive expression through the metallothionein promoter.
  • the genetic constructs also includes a transcription termination region.
  • Useful termination regions include a polyadenylation signal and the 3 ' -end of the gene from which the promoter region of the genetic construct was derived.
  • Other useful transcription termination regions include termination regions which are known to affect mRNA stability, such as those derived from the bovine growth hormone gene, globin genes, or the SV40 early region.
  • the linear or circular genetic construct includes an intron which can increase the level of expression of the heterologous gene.
  • the intron should be placed between the transcription initiation site and the translational start codon; 3' of the translational stop codon; or within the coding region of the gene encoding the lysostaphin analogs.
  • the intron should include a 5' splice site (i.e., a donor site), a 3' splice site (i.e., an acceptor site), and preferably includes at least 50 nucleotides between the two sites.
  • Particularly useful introns are those which are naturally found in genes of ruminants (e.g., genes encoding caseins).
  • Useful lipids include cationic liposomes, LIPOFECTAMINETM, LIPOFECTINETM, and other combinations of the lipids in appropriate ratios, as determined by the ability of the lipid(s) to help transfer a genetic construct into a cell.
  • Other useful DNA delivery systems are polyanions, polyamines, amphiphilic compounds, and solid coated particle delivered at high velocity with a gene gun.
  • bovine genomic DNA has been purified to obtain the gene encoding for the 1.5 kbp si-casein signal peptide by using the PCR amplification technique.
  • Two grams of bovine muscle were digested in a solution containing proteinase K, overnight at 55°C. Proteins were removed from the tube by washing with equal volumes of a phenol/chloroform mixture and the genomic DNA was purified by ethanol precipitation, allowed to dry and resuspended in a Tris (10 mM)-EDTA (O.lmM) solution.
  • Staphylococcus simulans biovar staphylolyticus (NRRL-2628) grown in CAA medium was harvested by centrifugation.
  • the bacteria were resuspended in 5 ml of 50 mM EDTA-50 mM Tris-HCl (pH 7.8) containing 50 ug/ml of lysostaphin (Sigma) and 0.5 mg/ml of lysozyme (Boehringer ) .
  • the suspension was incubated at 37°C for 2 h.
  • the bacterial DNA was washed with a phenol/chloroform solution and purified by ethanol precipitation .
  • the genes encoding for either bovine ⁇ s ⁇ -casein signal peptide or lysostaphin were specifically amplified using PCR technique. Amplification of the ⁇ si-casein signal peptide was performed by using oli- gonucleotide primers: bov-15'-ATC-CTG-CAG-TCT-GCC-ATC-ACC-TTG-ATC-ATC-3' (SEQ ID NO:1) bov-25'-CAC-GAT-ATC-GGC-AAG-AGC-AAC-AGC-CAC-AAG-ACA-3' (SEQ ID NO:2) To have a good processing of the lysostaphin mRNA into the eukaryotic cells, an ATG start codon was introduced into the 5 ' extremity of the lysostaphin gene to replace the natural TTG codon.
  • Lysostaphin gene was amplified with oligonucleotide primers: lys-15'-ACG-GAT-ATC-TCG-CGA-ATG-AGA-GCT-ACA-CAT-GAA-CAT-TCA-GC-3' (SEQ ID NO:3) lys-2 S'-AAG-ATA-TCT-CGC-GAT-CAC-TTT-ATA-GTT-CCC-CAA-S' (SEQ ID NO:4)
  • telomeres The initial cloning of PCR products was carried out using a pBR322 plasmid (New England Biolabs, Bev- erly, MA) . Cloned fragments were than introduced into pCMV-hGH (a home-made plasmid) plasmid in which the hGH gene was previously removed, and which includes the human early cytomegalovirus promoter and the bovine growth hormone polyA tail.
  • the new constructs form the pCMV-lyso-1 plasmid (Fig. 1) as eukaryotic expression vector. E. coli DH5 ⁇ was used as the host bacteria.
  • oligonucleotide primers were synthesized for use in in vi tro mutagenesis :
  • the underlined codons show the mismatched regions where the amino acids indicated in brackets replace the wild-type amino acids.
  • (Gly 123 ), (Glu 124) and (Gly 125 ) lysostaphin were constructed to remove a first N-glycosylation site at position 123 to 125 of the amino acid sequence of the mature form of the lysostaphin.
  • (Gly 23 ⁇ ), (Arg 231 ) and (Gly 232 ) lysostaphin were constructed to remove a first N-glycosylation site at position 230 to 232 of the amino acid sequence of the mature form of the lysostaphin.
  • EXAMPLE II Expression of constructs in a cell-free system
  • the genetic lysostaphin analog constructs were tested in a cell-free transcription and translation eukaryotic system. Transcription and translation were carried out with TNTTM Lysate Coupled Transcription/Translation Systems by Promega.
  • the DNA was mixed with lysate, reaction buffer, methionine-free amino acid mixture, RNasin, RNA polymerase and S 35 - methionine in the presence or absence of canine microsomal membranes for 90 minutes.
  • the results were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography .
  • Microsomal membranes were used to process signal peptide cleavage, membrane insertion, translocation and glycosylation (Fig. 3).
  • lysostaphin expressed with microsomal membranes has a higher molecular weight than lysostaphin expressed without membranes (lyso). Lysostaphin is appeared modified by glycolysation and this explain the results obtained with eukaryotic cells. In fact, the molecular weight of the modified lysostaphin was approximately 5 kDa higher than the non post-translationally molecule (Fig. 2). This may explain the lack of activity from lysostaphin produced in eukaryotic cells.
  • lysostaphin is glycosylated by eukaryotic cells
  • COS-7 transfected mammalian
  • endoglucanase PNGase Fl an enzyme which removes sugar molecules on N-glycosylated sites.
  • endoglucanase PNGase Fl an enzyme which removes sugar molecules on N-glycosylated sites.
  • endoglucanase PNGase Fl an enzyme which removes sugar molecules on N-glycosylated sites.
  • the molecular weight of endoglucanase-treated lysostaphin is comparable to commercially available lysostaphin.
  • Samples were loaded in the polyacrylamide gel as following:
  • Lane 3 and 4 5 ng of commercial recombinant lysostaphin, with (lane 4) or without (lane 3) PNGase Fl.
  • Lane 5 and 6 Supernatant of non-transfected cells with ( lane 6 ) or without ( lane 5 ) PNGaseFl.
  • New constructs (clone 5) with the Gly 125 - lysostaphin gene allowed for the production of an active and unmodified lysostaphin in the media of transformed cell lines in vi tro (Fig. 5).
  • Lane 3 Supernatant of transfected cells with human growth hormone (hGH) was used as a negative control.
  • Lane 4 Supernatant of Gly 125 -lysostaphin analog producing transfected COS-7 cells.
  • Lane 5 Supernatant of Gly 232 -lysostaphin analog producing transfected COS-7 cells.
  • Lane 6 Supernatant of Gly 125 Gly 232 -lysostaphin analog producing transfected COS-7 cells.
  • Lane 2 and 5 Translation analysis of lysostaphin mutated at position 127 with (lane 5) or without (lane 2) microsomal membrane.
  • Lane 3 and 6 Translation analysis of lysostaphin mutated at position 234 with (lane 6) or without (lane 3) microsomal membrane.
  • the activity of the recombinant Gly 125 - lysostaphin, Gly 2 2 -lysostaphin, and Gly 125 Gly 232 - lysostaphin analogs produced by CaCl2 transfected HC-11 (mouse mammary epithelial cells) cells was measured after 48 hours of in vi tro culture. 50 ml of the culture media to a S . aureus saturated agarose gel or by migration in a S. aureus saturated polyacrylamide gel. In both cases, a clear zone showing lytic activity of the enzyme was seen in the gel.
  • Fig. 7 After migrating culture media samples in a S. aureus saturated polyacrylamide gel, we can observe in Fig. 7 that when produced by HC-11 cells lysostaphin analogs are active, while the native form is not active.
  • the gel was loaded as following: Lane 1: Native mature lysostaphin construct Lane 2 : Recombinant Gly 125 -lysostaphin Lane 3: Recombinant Gly 23 -lysostaphin Lane 4 : Recombinant Gly 125-232 -lysostaphin
  • Lane 7 Culture media of recombinant hGH producing cells
  • the objective of this experiment was to demonstrate the ability of the plasmid constructs to produce an active recombinant lysostaphin after introduction of these constructs into mouse mammary glands .
  • mice Six week old Balb/c mice were obtained from Charles River Laboratories. Two, 5 and 10 ⁇ g of three selected DNA constructs were mixed with 1 ⁇ l of cationic liposomes in polystyrene tubes containing 50 ⁇ L of sterile phosphate buffered saline (PBS) and kept at room temperature for 30 to 40 minutes before injection.
  • PBS sterile phosphate buffered saline
  • mice were anesthetized with AVERTINTM (0.5mL/10g). The abdomen was thoroughly washed with 70% ethanol then dried. DNA-liposome mixtures were injected directly into the mammary tissue using 29G - 5/8 syringe-needle.
  • lysostaphin was not completely degraded in the mammary gland.
  • 10 ⁇ g of recombinant lysostaphin was injected into gland #1 (gland 1 left in front) one hour prior to tissue extraction.
  • the mice were sacrificed and the mammary glands were aseptically removed then homogenized in 1.0 mL of PBS with a POLYTRONTM homogenizer.
  • a 0.5 ml sample of each homogenate was kept to evaluate detectable activity of recombinant lysostaphin as previously described.
  • Cell wall lytic activity was analyzed on an agarose plate and acrylamide gel with heat-inactivated S . aureus (O.D. 2.0 at 600 nm).
  • RNA extraction small segments (200 to 300 ug) of the mouse mammary gland were treated with TRIZOLTM reagent (Gibco BRL) to extract total RNA.
  • TRIZOLTM reagent Gibco BRL
  • an extensive DNase treatment was done on all tissue biopsies.
  • the first DNA strand was then synthesized using a reverse transcriptase and an oligo-dT primer.
  • Polymerase chain reaction (PCR) using two lysostaphin gene specific primers, revealed any traces of messenger RNA in the tissue.
  • Fig. 8 shows that specific Gly 125 -lysostaphin analog mRNA was present in glands 2 (gland right in front), 3 and 7.
  • lyso-plates containing S . aureus ) received 25 ul of each gland extract to determine the presence of active lysostaphin in the tissue. The results showed activity in extracts from glands 1 and 2. The latest analyses were performed using SDS-

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Abstract

La présente invention a trait à des analogues de lysostaphine possédant une structure moléculaire empêchant les modifications intervenant après la traduction, tout en maintenant l'activité biologique du type lysostaphine. De tels analogues recombinants de lysostaphine présentent au moins un site modifié, ou un réagencement d'au moins un site au niveau de la séquence d'acides aminés de la protéine mature de lysostaphine. Cette invention concerne également des séquences d'ADN codant ces analogues de lysostaphine, ainsi que des plasmides recombinants et des cellules hôtes permettant d'exprimer un analogue de lysostaphine recombinant.
PCT/CA1998/000679 1997-07-23 1998-07-10 Analogues de lysostaphine recombinants WO1999005289A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002305624A CA2305624A1 (fr) 1997-07-23 1998-07-10 Analogues de lysostaphine recombinants
NZ502451A NZ502451A (en) 1997-07-23 1998-07-10 Recombinant lysostaphin analogs with amino acid substitution from Thr to Gly
EP98933404A EP0998570A1 (fr) 1997-07-23 1998-07-10 Analogues de lysostaphine recombinants
AU83285/98A AU734683B2 (en) 1997-07-23 1998-07-10 Recombinant lysostaphin analogs

Applications Claiming Priority (2)

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CA 2211293 CA2211293A1 (fr) 1997-07-23 1997-07-23 Analogues de lysostaphine recombinants
CA2,211,293 1997-07-23

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WO1999005289A1 true WO1999005289A1 (fr) 1999-02-04

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AU (1) AU734683B2 (fr)
CA (1) CA2211293A1 (fr)
NZ (1) NZ502451A (fr)
WO (1) WO1999005289A1 (fr)

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EP1224271A1 (fr) * 1999-10-19 2002-07-24 Bharat Biotech International, Ltd. Expression de la lysostaphine mure de recombinaison
WO2003082184A3 (fr) * 2001-12-21 2004-07-22 Biosynexus Inc Molecule de lysostaphine tronquee a activite staphylolytique amelioree
EP1463408A2 (fr) * 2001-12-21 2004-10-06 Biosynexus Incorporated Methodes et formulations visant a eradiquer ou a soulager la colonisation nasale staphylococcique, dans lesquelles est utilisee la lysostaphine
US6875903B2 (en) 1998-06-22 2005-04-05 University Of Vermont Treatment of Staphylococcus infections
US7091332B1 (en) 1998-06-22 2006-08-15 University Of Vermont Treatment of staphylococcus infections
CN102676559A (zh) * 2011-11-30 2012-09-19 西北农林科技大学 一种重组溶葡萄球菌素位点特异性整合载体及其构建的重组细胞
WO2015175774A1 (fr) 2014-05-14 2015-11-19 Trustees Of Dartmouth College Lysostaphine désimmunisée et méthodes d'utilisation

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CN102533821B (zh) * 2011-12-12 2013-06-05 西北农林科技大学 一种重组溶葡萄球菌素基因及其构建的表达载体和重组细胞

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WILLIAMSON C M ET AL: "EXPRESSION OF THE LYSOSTAPHIN GENE OF STAPHYLOCOCCUS SIMULANS IN A EUKARYOTIC SYSTEM", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 60, no. 3, 1 March 1994 (1994-03-01), pages 771 - 776, XP000579799 *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7091332B1 (en) 1998-06-22 2006-08-15 University Of Vermont Treatment of staphylococcus infections
US6875903B2 (en) 1998-06-22 2005-04-05 University Of Vermont Treatment of Staphylococcus infections
US6897041B1 (en) 1999-10-19 2005-05-24 Bharat Biotech International Limited Expression of recombinant mature lysostaphin
EP1224271A4 (fr) * 1999-10-19 2002-11-27 Bharat Biotech International L Expression de la lysostaphine mure de recombinaison
JP2003512050A (ja) * 1999-10-19 2003-04-02 バラット バイオテック インターナショナル リミテッド 組換え成熟型リソフスタフィンの発現
EP1224271A1 (fr) * 1999-10-19 2002-07-24 Bharat Biotech International, Ltd. Expression de la lysostaphine mure de recombinaison
AU784354B2 (en) * 1999-10-19 2006-03-16 Bharat Biotech International, Ltd. Expression of recombinant mature lysostaphin
EP1465990A4 (fr) * 2001-12-21 2006-06-28 Biosynexus Inc Molecule de lysostaphine tronquee a activite staphylolytique amelioree
EP1463408A4 (fr) * 2001-12-21 2005-12-07 Biosynexus Inc Methodes et formulations visant a eradiquer ou a soulager la colonisation nasale staphylococcique, dans lesquelles est utilisee la lysostaphine
EP1465990A2 (fr) * 2001-12-21 2004-10-13 Biosynexus Incorporated Molecule de lysostaphine tronquee a activite staphylolytique amelioree
EP1463408A2 (fr) * 2001-12-21 2004-10-06 Biosynexus Incorporated Methodes et formulations visant a eradiquer ou a soulager la colonisation nasale staphylococcique, dans lesquelles est utilisee la lysostaphine
WO2003082184A3 (fr) * 2001-12-21 2004-07-22 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
CN102676559B (zh) * 2011-11-30 2013-10-23 西北农林科技大学 一种重组溶葡萄球菌素位点特异性整合载体及其构建的重组细胞
CN102676559A (zh) * 2011-11-30 2012-09-19 西北农林科技大学 一种重组溶葡萄球菌素位点特异性整合载体及其构建的重组细胞
WO2015175774A1 (fr) 2014-05-14 2015-11-19 Trustees Of Dartmouth College Lysostaphine désimmunisée et méthodes d'utilisation
CN106795506A (zh) * 2014-05-14 2017-05-31 达特茅斯学院理事会 去免疫化溶葡萄球菌酶和使用方法
JP2017517278A (ja) * 2014-05-14 2017-06-29 トラスティーズ・オブ・ダートマス・カレッジ 脱免疫化リゾスタフィン及び使用方法
EP3143137A4 (fr) * 2014-05-14 2017-11-15 Trustees of Dartmouth College Lysostaphine désimmunisée et méthodes d'utilisation
JP2020168013A (ja) * 2014-05-14 2020-10-15 トラスティーズ・オブ・ダートマス・カレッジ 脱免疫化リゾスタフィン及び使用方法
CN106795506B (zh) * 2014-05-14 2020-11-27 达特茅斯学院理事会 去免疫化溶葡萄球菌酶和使用方法
US11091749B2 (en) 2014-05-14 2021-08-17 Trustees Of Dartmouth College Deimmunized lysostaphin and methods of use
US12104186B2 (en) 2014-05-14 2024-10-01 Insmed Incorporated Deimmunized lysostaphin and methods of use

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EP0998570A1 (fr) 2000-05-10
AU734683B2 (en) 2001-06-21
CA2211293A1 (fr) 1999-01-23
AU8328598A (en) 1999-02-16
NZ502451A (en) 2002-02-01

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