EP0998570A1 - Recombinant lysostaphin analogs - Google Patents

Recombinant lysostaphin analogs

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Publication number
EP0998570A1
EP0998570A1 EP98933404A EP98933404A EP0998570A1 EP 0998570 A1 EP0998570 A1 EP 0998570A1 EP 98933404 A EP98933404 A EP 98933404A EP 98933404 A EP98933404 A EP 98933404A EP 0998570 A1 EP0998570 A1 EP 0998570A1
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EP
European Patent Office
Prior art keywords
lysostaphin
gly
analog
recombinant
genetic construct
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98933404A
Other languages
German (de)
French (fr)
Inventor
Marc Gagne
Pierre Chapdelaine
Jacinthe Therrien
Dominic Gagne
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
IMMUNOPHARMA Inc
Original Assignee
Immunova Ltd
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Publication date
Application filed by Immunova Ltd filed Critical Immunova Ltd
Publication of EP0998570A1 publication Critical patent/EP0998570A1/en
Withdrawn legal-status Critical Current

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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

The present 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.

Description

RECOMBINANT LYSOSTAPHIN ANALOGS
BACKGROUND OF THE INVENTION
( a) Field of the Invention 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.
(b) Description of Prior Art
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. For example, 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. Sci. USA. (1987) 84:1127- 1131). The enzyme is synthesized as a proenzyme (molecular mass 59.0 kDa); its conversion to complete enzyme proceeds extracellularly (Recsei et al . , Proc. Nat. Acad. Sci. USA. (1987) 84:1127-1131). Previous studies have shown that 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. However, 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. In particular, the primary structure of a protein (i.e. its amino acid sequence) provides information that allows the formation of secondary (e.g. α-helix or β-sheet). It is well known that many cell surface and secretory proteins produced by eukaryotic cells are post-translationally modified, with one or more oligosaccharide groups for example. These 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. In fact, 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.
Inversely, 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. For example, 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 . In the 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.
It would be highly desirable to be provided with modified forms of 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.
SUMMARY OF THE INVENTION
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. Also recombinant plasmids are described 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. Also 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 .
In accordance with the present invention there is provided 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:
• a threonine residue substituted at position 125 of the amino acid sequence of mature lysostaphin;
• an asparagine residue substituted at position 123 of the amino acid sequence of mature lysostaphin; • a serine residue substituted at position 124 of the amino acid sequence of mature lysostaphin. The recombinant lysostaphin analog of the present invention include, without limitation, the following : Gly123-lysostaphin; Glux2 -lysostaphin .
Gly12^-lysostaphin;
Gly230_lysostaphin; Arg23 -lysostaphin;
Gly23 -lysostaphin;
Gly123Glu124-lysostaphin;
Gly123Gly125-lysostaphin;
Glu124Gly125-lysostaphin; Gly123Gly230-lysostaρhin;
Gly123Arg23x-lysostaphin;
Glyx23Gly23 -lysostaphin;
Glu124Gly230-lysostaphin;
Glul24Arg231_lysostaphin; Glu124Gly232-lysostaphin;
Gly125Gly230-lysostaρhin;
Glyx25Arg23x-lysostaphin; and
Gly125Gly232 -lysostaphin.
In accordance with the present invention there is also provided 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. Further, in accordance with the present invention there is also provided 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.
Furthermore, in accordance with the present invention there is provided 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.
In accordance with the present invention there is provided an in vivo method of producing a recombinant lysostaphin analog of the present invention in the milk of a ruminant mammal, the method 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:
• 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;
• forming a complex between the genetic construct and a lipid, the lipid may be cationic;
• forming a complex between the genetic construct and a polyamine;
• forming a complex between the genetic construct and an amphiphylic compound; and • any of these treatments of the genetic construct including cholesterol.
BRIEF DESCRIPTION OF THE DRAWINGS
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; and
Fig. 9 illustrates Staphylococcus aureus saturated polyacrylamide gel showing the activity of the recombinant lysostaphin analog produced in vivo into transfected mouse mammary glands.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, we have found that infusion of a genetic construct encoding lysostaphin analogs in in vi tro cultured eukaryotic cells, or into mammary glands of mammals, results in production of the lysostaphin analogs gene products in the mammary gland of the mammal. Accordingly, 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.
In various embodiments, the mammal is bovine, a sheep, a goat, or a pig. If desired, 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. For example, the analogs of modified gene derived lysostaphin are different than the native mature lysostaphin produced by Staphylococcus simulans or than - li ¬
the recombinant form of the mature lysostaphin produced by different genetically transformed prokaryotic microorganisms. In a preferred embodiment, 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. The introduction of mutations or nucleic acids changing into the DNA sequence of the mature lysostaphin gene allow to restore the activity of the corresponding lysostaphin analog when produced into eukaryotic cells. 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, in particular, 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. By "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. Alternatively, 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 .
In various embodiments of the invention, the transferred DNA is protected against degradation and the efficiency of gene transfer is increased by complexing the DNA with a DNA delivery system.
By "delivery system" is meant 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.
By "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. By "ductal tree" is meant the branched network of tubular structures which conduct milk in a mammary gland.
By "streak canal" is meant the papillary duct at the lower end of the teat which leads to the ductal tree
By "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 By "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. By "promoters" 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 51 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. Examples of 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. Other useful promoters include those which naturally drive the expression of mammary-specific genes. For example, the αsi-casein promoters, αs2~casein promoters, β-casein promoters, κ-casein promoters, β-lactoglobulin promoters, whey acidic protein promoters, and α- lactalbumin promoters can be used. If desired, 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. Examples of inducible promoters with steroid hormone-responsive elements, 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. During hormonal induction of lactation, the use of corticosteroids (e.g. dexamethasone) induces transcription through the MMTV promoter. The addition of zinc or cadmium to the ruminant's feed or water will drive expression through the metallothionein promoter.
Preferably, the genetic constructs (i.e., plasmid) 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.
Optionally, the linear or circular genetic construct includes an intron which can increase the level of expression of the heterologous gene. Generally, 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).
Several in vi tro and in vivo transfection systems can be used to enhance delivery into eukaryotic cells, most particularly mammary epithelial cells. Useful lipids include cationic liposomes, LIPOFECTAMINE™, LIPOFECTINE™, 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.
The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
EXAMPLE I Construction of pCMV-lyso-1
Using current molecular techniques, 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 αsχ-casein signal peptide or lysostaphin were specifically amplified using PCR technique. Amplification of the αsi-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)
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 DH5α was used as the host bacteria.
Mutagenesis of the lysostaphin gene
The locations of existing carbohydrate attach- ment sites within the lysostaphin amino acid sequence are shown in Fig. 2. The following oligonucleotide primers were synthesized for use in in vi tro mutagenesis :
(Gly123) lyso: 5'-TCA-TTT-TCA-GGC-TCA-GGT-GCC-CAA-GAT-CCA-ATG-CC-3' (SEQ ID NO:5)
(Glu124) lyso: 5'-TCA-TTT-TCA-AAT-GAA-GGT-GCC-CAA-GAT-CCA-ATG-CC-3' (SEQ ID Mι O:6)
(Gly125) lyso: 5'-TCA-TTT-TCA-AAT-TCA-GGC-GCC-CAA-GAT-CCA-ATG-CC-3' (SEQ ID NO:7) (Gly230) lyso: 5'-G-CCT-GTA-AGA-ACA-TGG-GGC-AAA-TCT-ACT-AAT-ACT-TTA-GG-3' (SEQ ID NO:8)
(Arg231 ) lyso: 5'-G-CCT-GTA-AGA-ACA-TGG-AAT-AGA-TCT-ACT-AAT-ACT-TTA-GG-3' (SEQ ID NO:9) (Gly232) lyso: 5'-G-CCT-GTA-AGA-ACA-TGG-AAT-AAA-GGC-ACT-AAT-ACT-TTA-GG-3' (SEQ IDNO:10)
The underlined codons show the mismatched regions where the amino acids indicated in brackets replace the wild-type amino acids. (Gly123), (Glu124) and (Gly125) 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. (Gly23^), (Arg231) and (Gly232) 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 TNT™ Lysate Coupled Transcription/Translation Systems by Promega. The DNA was mixed with lysate, reaction buffer, methionine-free amino acid mixture, RNasin, RNA polymerase and S35- 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).
As shown in Fig. 3, lysostaphin expressed with microsomal membranes (lyso + mb) 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.
EXAMPLE III
Effects of PNGase Fl on the molecular weight of recombinant lysostaphin
To verify whether lysostaphin is glycosylated by eukaryotic cells, a culture media of transfected mammalian (COS-7) cells was treated with endoglucanase PNGase Fl, an enzyme which removes sugar molecules on N-glycosylated sites. In vitro cultured cells were transfected by CaCl2 precipitation method described in the literature. As demonstrated in Fig. 4, the molecular weight of endoglucanase-treated lysostaphin is comparable to commercially available lysostaphin. Samples were loaded in the polyacrylamide gel as following:
Lane 1 and 2: Supernatant of transfected cells with mature lysostaphin construct with (lane
2) or without PNGase Fl (lane 1). 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. EXAMPLE IV
Production of recombinant lysostaphin analogs in In vitro cultured mammalian cells
New constructs (clone 5) with the Gly125- lysostaphin gene allowed for the production of an active and unmodified lysostaphin in the media of transformed cell lines in vi tro (Fig. 5).
Western blot analysis of analog and native mature lysostaphin was prepared as following: Lane 1: 5 ng of commercial recombinant lysostaphin was used as a positive control.
Lane 2: Supernatant of non-transfected
Lane 3 : Supernatant of transfected cells with human growth hormone (hGH) was used as a negative control.
Lane 4: Supernatant of Gly125-lysostaphin analog producing transfected COS-7 cells.
Lane 5: Supernatant of Gly232-lysostaphin analog producing transfected COS-7 cells. Lane 6: Supernatant of Gly125Gly232-lysostaphin analog producing transfected COS-7 cells.
Production of recombinant lysostaphin analogs in in vitro cell-free transcription-translation system
Mutated lysostaphin was also tested with TNT™ Lysate Coupled Transcription/Translation Systems (Fig.
6) as described in Example 2.
In Fig. 6, translation of the native lysostaphin construct and of the lysostaphin analog constructs, we can observe the following: Lane 1 and 4: Translation analysis of mature lysostaphin with (lane 4) or without
(lane 1) microsomal membrane.
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.
EXAMPLE V
Production of recombinant lysostaphin analogs by mammary gland epithelial cells
The activity of the recombinant Gly125- lysostaphin, Gly2 2-lysostaphin, and Gly125Gly232- 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.
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 Gly125-lysostaphin Lane 3: Recombinant Gly23 -lysostaphin Lane 4 : Recombinant Gly125-232-lysostaphin
Lane 5: Culture media of non-transfected cells
Lane 6: 5 ng of commercial recombinant lysostaphin
Lane 7: Culture media of recombinant hGH producing cells
EXAMPLE VI
In vivo assays in mouse mammary glands
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 . Experimental design
Six week old Balb/c mice were obtained from Charles River Laboratories. Two, 5 and 10 μg of three selected DNA constructs were mixed with 1 μl of cationic liposomes in polystyrene tubes containing 50 μ L of sterile phosphate buffered saline (PBS) and kept at room temperature for 30 to 40 minutes before injection.
Mice were anesthetized with AVERTIN™ (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.
To ensure that lysostaphin was not completely degraded in the mammary gland, 10 μg of recombinant lysostaphin was injected into gland #1 (gland 1 left in front) one hour prior to tissue extraction. Forty eight hours after injection of the constructs, the mice were sacrificed and the mammary glands were aseptically removed then homogenized in 1.0 mL of PBS with a POLYTRON™ 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).
Simultaneous with the tissue extraction, small segments (200 to 300 ug) of the mouse mammary gland were treated with TRIZOL™ reagent (Gibco BRL) to extract total RNA. To eliminate the risk of contamination with plasmidic construct DNA, 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 Gly125-lysostaphin analog mRNA was present in glands 2 (gland right in front), 3 and 7. For qualitative analysis, 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-
PAGE without sample denaturation and containing heat- killed S. aureus . Bacteriolytic activity of the intramammary produced recombinant lysostaphin was clearly shown in this experiment. The recombinant enzyme from mouse mammary glands was shown having an adequate molecular weight (Fig. 9).
In conclusion, it appears from the present assays that it is possible to perform intramammary injections of plasmid DNA constructs for production of recombinant antimicrobial proteins.
The disclosure of all patents, publications, including published patent applications, database accession numbers, and depository accession numbers referenced in this specification are specifically incorporated herein by reference in their entirety to the same extent as if each such individual patent, publication, database accession number, and depository accession number were specifically and individually indicated to be incorporated by reference in its entirety.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any varia- tions, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims .

Claims

WE CLAIM :
1. 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 said modified site is at least one added, changed, substituted, or deleted amino acid residue.
2. The recombinant lysostaphin analog of claim 1, wherein the post-translational modification is glycolysation, methyla ion, disulfide bond formation, acetylation, phosphorylation, or sialylation.
3. The recombinant lysostaphin analog of claim 2 wherein the modified site is for improving the lysostaphin-like biological activity after production by eukaryotic cells.
4. The recombinant lysostaphin analog of claim 2, wherein the modified site is a threonine residue substituted at position 125 of the amino acid sequence of mature lysostaphin.
5. The recombinant lysostaphin analog of claim 2, wherein the modified site is an asparagine residue substituted at position 123 of the amino acid sequence of mature lysostaphin.
6. The recombinant lysostaphin analog of claim 2, wherein the modified site is a serine residue substituted at position 124 of the amino acid sequence of mature lysostaphin.
7. The recombinant lysostaphin analog of claim 1 is selecteidd ffrroomm tthhee ggrroouupp ccoonnssiissttiinngg of: Gly123 -lysostaphin; Glu124 -lysostaphin; Gly125 - lysostaphin; Gly23┬░- lysostaphin; Arg23 x - lysostaphin; Gly232 -lysostaphin; Gly1 3Glu124 -lysostaphin Gly123Gly125-lysostaphin Glu124Gly125-lysostaphin Gly123Gly230-lysostaphin Gly123Arg231-lysostaphin, Gly123Gly232 -lysostaphin Glu124Gly23 ┬░ - lysostaphin Glu124 rg23 x - lysostaphin Glu1 4Gly232-lysostaphin,- Gly125Gly23┬░-lysostaphin; Gly125Arg231-lysostaphin; and Gly125Gly232 -lysostaphin.
8. A DNA sequence encoding the recombinant lysostaphin analog of claim 1, having substantially an nucleic acid sequence encoding mature lysostaphin which includes at least one modified site, and wherein said modified site is a nucleic acid addition, changing, substitution, or deletion.
9. A DNA sequence encoding recombinant lysostaphin analog according to claims 4 to 7.
10. A DNA sequence encoding a recombinant lysostaphin analog having substantially an nucleic acid sequence encoding mature lysostaphin which includes a rearrangement of at least one site for inhibiting post- translational modifications.
11. A therapeutic composition comprising a therapeutically effective amount of one recombinant lysostaphin analog or of a mixture of more than one analog according to any of claims 1 to 7 in association with a pharmaceutically acceptable carrier.
12. A method of producing a recombinant lysostaphin analog according to any of claims 1 to 7, which comprises the steps of: a) transfecting an eukaryotic cells with an inducible expression vector comprising a coding DNA sequence of claims 8 to 10 and a signal peptide; b) inducing said expression of the coding DNA sequence to produce the recombinant lysostaphin analog.
13. The method of claim 12, wherein the eukaryotic cells are mammalian cells.
14. The method of claim 13, wherein the lysostaphin analog is produced in vi tro .
15. The method of claim 13, wherein the lysostaphin analog is produced in vivo .
16. An in vivo method of producing a recombinant lysostaphin analog of claims 1 to 7 in the milk of a ruminant mammal, said method comprising the steps of: a) preparing a genetic construct including a DNA sequence encoding said recombinant lysostaphin analog of claims 1 to 7 and a signal peptide in a liquid carrier to form a liquid complex, b) infusing said liquid complex into a cistern, ductal tree, and/or alveoli of a mammary gland of the ruminant mammal and allowing transfection of said mammary gland to permit expression and secretion of the lysostaphin analog into the milk of said mammal ; c) collecting milk from said mammal; and d) purifying the lysostaphin analog from the collected milk.
17. The method of claim 16, wherein said mammal is a bovine .
18. The method of claim 16, wherein said mammal is a sheep .
19. The method of claim 16, wherein said mammal is a goat .
20. The method of claim 16, wherein said mammal is a porcine
21. The method of claims 16 to 20, wherein said lysostaphin analog is enzymatically active on staphylococci .
22. The method of claim 16 wherein said genetic construct is treated to enhance its ability to cross the membrane of an epithelial cell.
23. The method of claim 22, wherein said treatment of said genetic construct comprises forming a complex between said genetic construct and a polycationic compound .
24. The method of claim 23, wherein said polycationic compound is selected from the group consisting of poly-lysine and poly-ornitine .
25. The method of claim 22, wherein said treatment of said genetic construct comprises forming a complex between said genetic construct and a lipid.
26. The method of claim 24, wherein said lipid is cationic .
27. The method of claim 22, wherein said treatment of said genetic construct comprises forming a complex between said genetic construct and a polyamine.
28. The method of claim 22, wherein said treatment of said genetic construct comprises forming a complex between said genetic construct and an amphiphylic compound .
29. The method of claim 22 to 27, wherein said treatment of said genetic construct includes cholesterol .
EP98933404A 1997-07-23 1998-07-10 Recombinant lysostaphin analogs Withdrawn EP0998570A1 (en)

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CN102676559B (en) * 2011-11-30 2013-10-23 西北农林科技大学 Recombinant lysostaphin locus specific integration vector and recombinant cell established by same
EP3143137A4 (en) * 2014-05-14 2017-11-15 Trustees of Dartmouth College Deimmunized lysostaphin and methods of use

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