AU734683B2 - Recombinant lysostaphin analogs - Google Patents
Recombinant lysostaphin analogs Download PDFInfo
- Publication number
- AU734683B2 AU734683B2 AU83285/98A AU8328598A AU734683B2 AU 734683 B2 AU734683 B2 AU 734683B2 AU 83285/98 A AU83285/98 A AU 83285/98A AU 8328598 A AU8328598 A AU 8328598A AU 734683 B2 AU734683 B2 AU 734683B2
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- AU
- Australia
- Prior art keywords
- lysostaphin
- analog
- vector
- recombinant
- set forth
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- C12N15/09—Recombinant DNA-technology
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Description
I, (1 WO 99/05289 1 PCT/CA98/00679 RECOMBINANT LYSOSTAPHIN ANALOGS BACKGROUND OF THE INVENTION Field of the Invention The invention relates to lysostaphin analogs having a molecular structure preventing posttranslational 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 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 LA I I LIt:.\ Wil *j 'J I- di 11 2? Ii .3ji +a8L2_ +W 811~ 2 U 4ti ZZ:Jb Uu1-$AIIRu WALI~bN MUSUCk +I I44U U~ 14U'u Ir-4302 -2 conditions coordinate hexosarnirxidase and thiolproteinase (Donham. et al.. J. Gen. Microbial, (1988) 134:(2615- 2621). The gene for lysastaphin-endopeptidase synthesis represents 1. 5 kbp (Recsei et al.. Proc. Nat Acad.
Sci. UISA. (1987) 84:1127-1133.) The enzyme iis synthesized an a proenzyme (molecular mass 59.0 kDa); its conversion to complete enzyme proceeds extracellularly (.Recsei et Proc. Nat. Acad. Sci.
USA. (1987) 84:1127-1131).
Previous atudie8 have shown that lysastaphin can he produced by fermentation techniques wherein S.
.sizulains is grown in liquid culture. Recombinant DNA techniques are well known, whereby genes for a variety of proteins can be cloned by inaertion into a cloning and expression vector, which can thern be introduced into microcrganisms for production of the same proteins unmder recombinant form. Microorganismsl, such as E. coll, Bacili~us and Streptomryces epp have been used to produced large amount of recombinant lysoetaphin.
However, there have been no reports with evident data relating to such cloning techniques being used to insert the genes encoding lyaostaphin into a cloning vector to reconstruct novel vectors which can transf ect euk.aryotic cells to allow the production of lysostaphin either in2 vitro or in vi.vc. These new vectors can provide new alternative to bacterial production systems of lysostaphini, or can be introduced In viZvo into an organ, likce mammary glands of commercial milk producing 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 prirrY structure of a protein its amino acid sequence) provides information that allows the formation of secondary a-helix or z:b Ua-' tIfIAU WAL~hR MSUCIES +I-14 i-4JbZ h-UZI F Ub/ I I rayIUON -3it i.5 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 S 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 1- Proteins can be included in the grcupr. Cf phosphcylaion, acetylation, methylation, or sialylation. Proper glycolylation can be essential for biological activity. In fact, some genes from eukaryotic organisms when expressed in bacteria S. col4) is which lack cellular processes for glycosylatinag proteins, yield proteins that are recovered with little or no activity by virtue or their lack of glycosylat ion.
Inversely, prokaryotic proteins, like 2C lysostaphin, which are naturally mpodified. followingr their translation into bacterial, even so can be altered by post -cranslational modifiLcations and become biologically inactive because of th~ese undesirable biochemical chan43ing.
For example, glycosylation occurs at speciraic locations along the polypeptiLde backbone and is usually of two t-voes: 0)-linked ol~sc~r~csare a ce to serine or threonirne 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 contain glycosylation sites ini its amino acid sequence could be rendered biologically inactiLve if post-translationally glycosylated in eukaryotic cells in vitro or in vivo.
AMENDED SHETj WO 99/05289 PCT/CA98/00679 4 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 vitro and in vivo systems are provided.
0 .J t jj. A- UO-WMEAAU WALKER~ ASSUMSk +I~4:'45 E-0Z1 F 1 .Ut/ TraYVIU The present invention provideo a solution to the problem of non-desired post -translational modifications of recombinant lyaostaphin 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 included at least one modified site for inhibiting post- translational modification after production by eukaryotic cells, wherein the modified site is at least one added, criange., oustituted, or deleted amino acid residue.
The post-translational modification is glycosylation, metbhylation, disulfide bond formation, acetylation, phosphorylatiol, or sialylation.
The modi.fied site of the recomrbinant lysostaphin analog is for improving the lysostaphin- like biolcgical activity after production by eukaryotic cells.
Such a modified site in accordance with the present invention may be one of the following: 0 a threonine residue substituted at Dosition 127 of the amino acid sequence of mature lysostaphin; an asparagine residue substituted at position 125 of the amino acid sequence of; mature lysostaphin; a serine residue substitutes at position 126 of the amino; acid sequence of mature lysostaphin.
The recombinant lysoataphin analog of the present invention include, without limitation, the following: AMENDED
SHEET
UI-I 1-0 V~ UG-MANKI I flI:AU WALAti iUili i h5h' 1' -5 4414M).3/Z I G1ya 5 lycataphin, GluL 26 .lyastaphin; Gly 1 7- lysostaphin; Glyl- lyecs taphin; Argw32lysostaphin; Oly 23 4 -lysaostaphin; Gly1lGlull" lysos taph in; Gjy12:"GlyI- 2 lysos t aphin; Glu'2"Gly 1 lyastaphin; Gly 2 5 Gy23- yastaphin; G:ly'2Ar 2 3 3 lysostapbhin; Gl4y 1 2 'Gly~l 4 lysostapb4.n; GlU 12 r~rly 2 JlySoetaphin; Glu -2Arg 2 l' lyeastaphin; CGjU-6l1- lyastaphin; G y12 7 rGl-1 2 lysostaphin; Gly -Ar9 3 2-lysostapiin; and Gly1 27 Gly 2 3 4 lysostaphin; :i accordance with the present invention there is also provided a DNA sequence encoding a recombinant lysostaphin- analcg, having substantially a nucleic acid sequence encoding mature lysostaphin which incoluded at least one modified site, and wherein the modified site is a nucleic acid addition, mutation, substitution, or deletion.
The DNA sequences of the present invention include, wizhout limitation, the nucleic ac,, sequences encoding the recombinant lysostaphin analogs of the present invention.
The DNA sequence encoding a recomnbinant lysostaphin analog having su..bstantially a nucleic acid sequence encoding mature lysostaphin may iniclude a rearrangement of at least one site for inhibiting posttranslational modifications.
AMENDED
SHEET
WO 99/05289 PCT/CA98/00679 7 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 vitro 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 WO 99/05289 PCT/CA98/00679 8 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 polyornitine; 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 WO 99/05289 PCT/CA98/00679 9 in vitro 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 vitro 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 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 vitro 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, U( XZ:J( UU-MANIM~ AU i~ A I- ri):a3++j b) mixing the construct with a delivery system to form a complex, C) infusing the complex into a culture wel~l containing eukaryotic cells, or into the mammary gland of the mammal, d) raising the cultured eukaryotic cells so that the lyastaphin analogs encoded by the construct is expressed and secreted into the culture medium, e) raising the mammal so that the lysostaphin analogn encoded by the constr-ucts are expreed and secreted into the milk produced by the secretory cells of the udder ductal tree and the mammary alveolar cells, f) obtaining the in vitr-o culture medium or the milk from the mammal. The invention also features a composition which included milk produced according to this method.
In variousa embodiments, the mammal is boviLne, a sh~eep, a goat, or a pig. Zf desired, a DNA delivery system complex can be infused into the mammal at any age of sexual maturity.
The subjectL invention provides mature lysostaphin analogs and the modified DNA sequence encoding for these lyaostaphin analogs. The present invention provided for recombinan~t plasinids which have been created by insertion of the 741 base pairs (hp) mutated DNA fragment encoding for the mnature form oZf the lysoataphin analogs. The plasrnids are cloning vectors that replicate in various host microorganisms, such as E. coli, and that allow transcription and translation of an active lysostaphin analogs both by in vitro and 1n vivo transfected eu)karyotic cells. The analogs of lysostaphin are different from the native mature lysostaphin produced by Staphylococcus siinulanS or from 7 ,Vt S. SS~~.SS.S 0~ a' Z- JUL L- 1 j JJi 4.JUf r- U UU 2:3JUUJ4.4#U5 A 7 the recombinant foarm of the matur~e 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 a fixed number greater than 0) of mutated (changed) nucleic acid residues per codon, for which correspond a lysostaphin peptide analogs having one or more amino acid residues changed compared to the original amino acid residues in the native molecule. The introduct~ion of mutacions or nucleic acids changing into the DNA sequence of the mature lysostaphin gene allows the restoration of the activity of the corresponding lysostaphin analog when produced into eukaryotic cello. The production .in vivo IS or in vitro of lycostaphin analogs by t'he corresponding mutated gene in transfected eukarycti-,c cells restore (give back) the enzymatic activity to the lysostaphi~n anialogs. The present, recombinant plasmids express lysastaphin analogs in% high levels in cloned transf acted mammalian cells harboring the plasmids. Lysostaphin analogs are produced and secreted by transfecced cells and accumulaces in vitro in large quantities in the medium in which the transfected cells are cultured.
The vresenit invention, in particular, provides for genetic constructs, which include any ubiquitous or inducible promoters active in eukaxyotic cells, a signal peptide working in eukaryotic cells, more particularly, in mammali.an cells, and the modified lyaostaphin gene.
By %signal peptideO is meant a polypeptide which facilitates secretion of the protein to which it is linked. The signal peptide can be naturally occurring in the lyeastaphi.n DNI~A sequence. Alternatively, the genetic construct can be engineered AMENDED S -A 1-9 II .I 1I tl -L ;Sul +;182- l-4. Up ~U 44~ V(-Ih MZJU U-MA(TINEAU WAL~kR ASSUCIE +I-WI4.dU1-438Z ki-U] 1 .I/ZI TFraYlug -12 so that a signal peptide is bonded to the lysootaphin analogs.- The invention provides also a convenient and efficient method for directly transferring the modified )ysostaphifl gene into the mammary gland of the ruminant ma~mmal to produce a lysootaphin 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 complexixu3 the DUA with a DNA delivery system.
By "delivery system" is meant cationic, neutral, negatively- charged, polycationic, pH-sensitive lipids, polyicn, amphiphilic compounds, and Polycatior-ic amino is poiymers, which can complex with the genetic constrcts to allow and/or enhance their ability to cross the membrane of the secretory cells mammary gland epithelial cells) in vivo or in vitro. These treatments can improve cell 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 str-eak canal.
By "ductal. treem is meant the branded network of tubular structures which conduct milk in a mammary gland.
By "streak canal" is meant the papillary duct at lower end of the teat which leads to the ductal tree.
By '4 secretory cells" is meant the epithelial cells of the ducts and alveoli of the mammary gland, or any other of the mammary able to secrete a recombinant lysostaphin analog into the milk.
AM I9EI SH.EET UII9 ze.3V Us-MAIMIlAU WALIUk A45UIEIS j'It;i U.+1-014-1-4401 EU I P I2/l ITravII -13 By Amnodified Seneo ia meant a del.etion, an insertion, an inversion, or a base substitution of at least one nucleic acid of the gene, and mutation of.r at least one amino acid residue of the lysostaphin.
S By "promaters"v 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' sequence naturally upstream of the lysostaphin analogs encoding region iz u3ed to refer to the 5, sequ.ence 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 th.e human cytomegalovirus (CMV) is immediate early promoter, the simian Virus 40 (SV4O), the Rous Sarcoma Virus (SRVI), the adenovirus major late promoter. Other useful promoters include those which naturally drive the expression of mammary-specific genes. For example, the aoi5-casein promoters, caS2-casein promoters, -casein promcters, K-casein promcters, 53lactoglobulin promoters, whey acidic protein promoters, and a-lactalbuain promoters can be used. if desired, the promoter can be operably linked to one or more enhancer elements such that the enhancer element(s) increases transcriptio-n of the gene encoding the lysostaphin analogs. Useful enhancer elements include, without limitation, enhancer elements from ICMV, S7,40, and the R.SV lcng terminal repeat. Expression of the lyaostaphin 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 hsp methods for inducing these promoters are described in AMENDED SHEET WO 99/05289 PCT/CA98/00679 14 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 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 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 splice site a donor site), a 3' splice site 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 genes encoding caseins).
Several in vitro and in vivo transfection systems can be used to enhance delivery into eukaryotic cells, most particularly mammary epithelial cells.
Useful lipids include cationic liposomes, LIPOFECTAMINETM, LIPOFECTINETM, and other combinations WO 99/05289 PCT/CA98/00679 15 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 aSl-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 0 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 (0.1mM) 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 ug/ml of lysostaphin (Sigma) and 0.5 mg/ml of lysozyme (Boehringer). The suspension was incubated at 37 0 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 aSl-casein signal peptide or lysostaphin were specifically amplified using PCR technique. Amplification of the U-I-s zz: V Us"MNI I RhAU WALI~kI R_ UL~f, 1 .f ctSl-casein signal peptide was performed by using oligonucleotide primers: boy-I. 5' -ATC-CTG-CAG-TCT-GC-C-ATC-ACC-?1G-ATC-ATC-3' (SEQ ID NO:1) bov-2 5' -CAC-GAT-ATC-GGCAAG-AGC-AAC-AGC-CAC-AAQ-ACA-3' (SEC ID NO: 2) To have a good procezoing of the Iyastaphin mRNA into the eukcaryotic cells, and AM start codon was introduced into the 5' extremity of the l1yeostaphin gene to rep.laetentrlTGcdn Lysostaphin gene was amplified withb oligonucleotide primners; lye-i 5'1 -ACCG-CAT -ATC-TC-CGA-&T_- -AG- CT ACA-CAT-G-AA- CAT -TCA-GC 3 (SEQ 11) NO:3) lys-2 51-AAG-ATA-TCT -CGC -GAT-CAC -TlT-ATA-GTT- CCC- CAA- 3' (SEQ ID) NO:4) The initial cloning of PCR products was carried out using a pBR322 plaamidl (New England Biola.bs, Beverly, is MA) cloned fragments were then introduced into pCMV-hGHi (a home-made plasmid) plasmid in which the hGIR gene was previously removed, and which included the human early cytomegalovirus promoter and the bovine growth hormone poJlyA tail. The new conztr,.cta fonrm the pCIV-lyso-a Plasmid (Fig. 1) as eukaryotic expression vector. E. coli was used as the host bacteria.
Mutagenesia of the lysostaphin gone The locations of existing carbohydrate attachment sitez within the lysostaphin. amino acid sequence are shown in Fi-g. 2. The following oligonucleamide primers were synthesized for use in in vitro mutagenesis: (Gly'2) lyc ~c-3 ~fsf, ID NO: (Glu: 26 lyso: 5' TCPL-tT-TCA-AAT -MA-GGT-GCZ-CAA-GA'- CCA-ATG-CC- 3' (SEQ MD NO: 6) lyso: 5' -TCA-T."-TCA-AAT-TCA-C-GCC-CAA-GAJ-CCA-ATG-CC-3' (SEQ ID NO -7) AMENDED
SHEET
U (11-Vj ZZ:4V We-MKOMNbA ALUM AS.AS -4 V. Ij j I j i -17- (Gly"O) ).ysc: S T-GA-AGA-A-TG-Q-AAA-TCT-AT-AAT -ACr-TIA-G3- 2' (SEQ MD NO: 8) (Axgl-Iz) Iyao: 5' 1 -GCC--AhCA-T- -AATW =ACr-AAT -ACT- TTA-GG -3' (SEQ ID NO: 9) 5(Gliy43) Iywo: 5' G-Ccr-GTA-AiDA-A A-'ZGG-AA -AAA-92-AT-AAT-AC-TA-3- 3' (SEQ ID b7O: The underlined codons show the mismatched regions where the amino acids indicated in brackets replace the wild-type amino acids.
(C-2y~r) ,((31U12) and (Gly" 7 1yeostaphin were constructed to remove a first N-glycosylation site at position 125 to 127 of the amino acid sequence of the mature form of the lyaostaphin. (Gly 2 32 (Arg 33 and (Gly23Th lyeostaphiz' were constructed to remave a second Nis glycoaylation site at position 232 and 234 of the amino ac~d sequence of the mature form of the lysostaphin, as shown in Figure 2.
Expression of conatru4cte in C. call-free eyatom The gener-ic lysostaphin analcg constructs were tested in a cell-free transcription and 'translation eukaryotic system. Transcription and translation were carried out with TNTm Lysate Couipled Tz'anacription/Translation Systems of P1-omega. The DNA was mixed wich lysate, reaction buffer, methionine- free amino acid mixture, R~asin, RNA pol.ymerase and S 3 6 -methionine in the presence or absence of canine rricroscmal memb;-ranes 'fzr minutes. The results were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) and autoradi4oaraphy. Microsomal membranes were used to process si.gnal 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 -7 411 MENOED SviEET
C)
WO 99/05289 PCT/CA98/00679 18 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.
This may explain the lack of activity from lysostaphin produced in eukaryotic cells.
EXAMPLE III Effects of PNGase F1 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 CaC12 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 Lane 3 and 4: Lane 5 and 6: mature lysostaphin construct with (lane 2) or without PNGase Fl (lane 1).
5 ng of commercial recombinant lysostaphin, with (lane 4) or without (lane 3) PNGase Fl.
Supernatant of non-transfected cells with (lane 6) or without (lane PNGaseFl.
U-1 I-MUR Ii UsI I IRhAU WAL'AkN' ASUCih L 4i J. u Production of recombinant -yoatapkdn analcgs In In v4tro cult~ured mamalian cells.
New constructs (clone 5) with the Gly 1 2 1 -lyscstaphin gene dllowed for the production of an active and unmodif ied lysostaphin in the media of transformed cell lines in vitrro (Fig. Western blot analysis of analog and native mature lypostaphin was prepared as follows: Lane 1: Supernatant of transfected cells with non-mcdified lysostaphin.
Lane 2: Supernatant of Gly 1 2'-lyscdstaphin analog producizng transfected COS-7 cells.
Lane 3: Supernatant of Gly',2Gly 23 4 -lysoetaphin analog 1s producing transfected COS-7 cells.
Lane 4: Supernatant o-6 Gly"I-lyscataphin analog prcdlucirng transfectad COS-7 cells.
Lane 5: Supernatanc of non-transfected (NT) cells.
Lane 6: 5 nc of ccrnmercial recombinant lysostaphin was used as a posimive control.
Ldne 7: Suveatant of transf soted cells with human growth Mormone (hGH) used as a negative contral.
Production of recombinant lysostapbhin analogs in in vi tro cell-free transcription- tram lation system mutated lyaostaphin was also tested with TNT"' Lysar-e Coupled Transcription/Transl1at ion Systems (Fig. 6) as described in Example 2.
LnFw. 6, tr-anslatiJon of the riative ytp~ccnstruct and of the lyaostaphin analog constructs, we can observe che following: Lane I. and 4: Translation analysi.s of lyaostaphi-Ln mutated at position 127 with (lane 5) or without (lane 2) micro somal membrane.
Lane 2 and Translation analysis of lysostaphin mutated at position 234 with (lane 6) or without (lane 3) microisamal memrbane.
Arr 0 uI~i ~~7z~4 UGftIAki IMEAU WALMkK ASSULIS I -t "I fM S)J.E U F-i 5M *;iJU-4i.),,j 20 Lane 3 and 6: Translation analysis of mature lysostaphin with (lane 4) or without (lane 1 microsomal membrane.
S ZZMAMPL...3 Production of recominant Ilymostaphin analogs by mamm~ary gland ep4thelial cells The activity of the recombinant Gly*ias lyscstaphjn, Gly 232 -lyscstaphin, and Gly'2 5 -Gly 232 lyscstaphin analogs produced by CaCl, transfecced HC-11 (mouse mammary epithelial cells) cells was measured after 48 hours of in vitro culture. 50 ml of the cult-ure media to a S. aureuz saturated agarose gel or by migration in a S. aureus saturated polyacryIlarnide ael. 7n both cases, a clear 2ofle showing lyt4c activity of the enzyme was seen in the gel.
After migrating culture media samples in. a S. auzeus satuz-atad polyacrylamide gel, we can observe in Fig. 7 that when produced by HC-1l cells lysostaphin analcgs are active, while the native for-m is not active. The gel was loaded as follows; Lane 1: Native mature lysostaphin construct Lane 2: Recombinant Gly 2 7 -lysostaphin Lan~e Recombinant Gly 2 3 4 -lysotaphin Lane 4: Recombinant Gly 1 2 7 -3 4 -1 lysost aphin Lane S: culture media of non-transfected cells Lane 6: 5 na of commercial recomrbinant lysostaphin Lane 7: Culture media of xrecombinant hGh Droducing cells i~n vlvo assays In mouse mamimary glands The objective of this experiment was to demonstrate the ability of the plasrnid conatructs to produce an active recombinant !ystaphin after introduction of these constructs into mouse mammary glands.
7 MENEO S1A
EU
WO 99/05289 PCT/CA98/00679 21 Experimental design Six week old Balb/c mice were obtained from Charles River Laboratories. Two, 5 and 10 gg of three selected DNA constructs were mixed with 1 4l of cationic liposomes in polystyrene tubes containing 50 p L of sterile phosphate buffered saline (PBS) and kept at room temperature for 30 to 40 minutes before injection.
Mice were anesthetized with AVERTINTM (0.5mL/10g). The abdomen was thoroughly washed with 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 p g 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 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 2.0 at 600 nm).
Simultaneous with the tissue extraction, small segments (200 to 300 ug) of the mouse mammary gland were treated with TRIZOLTM 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 LIA .11. A .L IL t 4' 1 L L i. .d 4) riU MjUI J UI4t~ 1 i!VV MZ40 Uu-WIJINEAU WALKERK ASSMLIkS I-4 4 "4iZt-UZI Vr 7(iu i Ivius -22gene apecific primers, revealed any traces of meesenger RNA in the tissue.
Fig. 8 shows that specific Gly'"-lysostaphin analog mRNA was present in glands 2 (gland right in front), 3, and 7. F'or qualitative analysis, lyso-plates (containing S. aureus) received 25 Asl 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 aureus. Bacteriolytic activity of the Intramammary produced recombinant lyaostaphin was clearly shown in this experiment. The recombinant enzyme from mouse mammary glands was shows having an adequate molecular weight (Fig. 9) in conclusion, it appears from the presen-c assays ,that it is possible to perform intramammary injeccions of plasmids DNA constructs for production of recombinant antimicrobial proteins.
The disclosure of all patents, publications, including published patent application, database accession numbers, and depository accession numbers referenced in this specification are specifically in-corporated herein by reference in their entirecy to the same extent as if each- such individual patent, publiction, database accession number, and depository accession number were specifically and .cu2y indicated to be incorporated by reference i.n i';ts entirety.
While the invention has been described in connection with specific embiodiments thereof, it will be understood that it is capable of further modifications and this applicat-ion is intended to cover any variations, uses, or adaptations of the invention following, AMENDED
SHEET
WO 99/05289 PCTICA98/00679 23 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.
EDITORIAL NOTE 83285/98 The sequence listing is numbered from page 1 4. The claims pages follow starting from page number 24.
WO 99/05289 PCT/CA98/00679 1 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: IMMUNOVA LTEE.
(ii) TITLE OF INVENTION: RECOMBINANT LYSOSTAPHIN ANALOGS (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: SWABEY OGILVY RENAULT STREET: 1981 McGill College Avenue Suite 1600 CITY: Montr6al STATE: QC COUNTRY: Canada ZIP: H3A 2Y3 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette COMPUTER: IBM Compatible OPERATING SYSTEM: Windows SOFTWARE: FastSEQ for Windows Version (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:
CLASSIFICATION:
(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: CA 2,211,293 FILING DATE: 23-JUL-1997 (viii) ATTORNEY/AGENT INFORMATION: NAME: C8te, France REGISTRATION NUMBER: 4166 REFERENCE/DOCKET NUMBER: 12898-11PCT FC/ld (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 514 845-7126 TELEFAX: 514 288-8389
TELEX:
INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA SUBSTITUTE SHEET (RULE 26) WO 99/05289 PCT/CA98/00679 2.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATCCTGCAGT CTGCCATCAC CTTGATCATC INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CACGATATCG GCAAGAGCAA CAGCCACAAG ACA 33 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ACGGATATCT CGCGAATGAG AGCTACACAT GAACATTCAG C 41 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: AAGATATCTC GCGATCACTT TATAGTTCCC CAA 33 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SUBSTITUTE SHEET (RULE 26) WO 99/05289 PCT/CA98/00679 3 (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID TCATTTTCAG GCTCAGGTGC CCAAGATCCA ATGCC INFORMATION FOR SEQ- ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: TCATTTTCAA ATGAAGGTGC CCAAGATCCA ATGCC INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TCATTTTCAA ATTCAGGCGC CCAAGATCCA ATGCC INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GCCTGTAAGA ACATGGGGCA AATCTACTAA TACTTTAGG 39 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs SUBSTITUTE SHEET (RULE 26) WO 99/05289 PCT/CA98/00679 4 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GCCTGTAAGA ACATGGAATA GATCTACTAA TACTTTAGG 39 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID GCCTGTAAGA ACATGGAATA AAGGCACTAA TACTTTAGG 39 SUBSTITUTE SHEET (RULE 26)
Claims (24)
1. A lysostaphin analog having substantially an amino acid sequence of native lymostaphin which includes a modified poet-t'ra.noldtional glycocylation eite at amino acid residues 22 to 127 or 232 to 234 of the amino acid sequence shown in Figure 2.
2. The lysostaphin analog of claim I wherein the modified site prevents N-glycosylation.
3. The lysostaphin analog of claim 2, wherein the modified site is a threonine, residue at position 127 of the native lyaostaphin which io changed for a glycine residue.
4. A nucleic acid encoding the lysostaphin analog of any one of claims 1 to 3.
5. A recombinanc vector comprising the nucleic acid of claim 4.
6. A recombinant vector as set forth in claim which is an expression vector.
7. A recombinant vector as set forth in claim 6 wrhich is an eukaryctic expression vector.
8. A recombinant vector as set forth in any one of claims 5 to 7 which further comprises a signal peptide capable of directing secretion of the 2yostaphin analog by a hoot cell.
9. A recombinant host comprising the recombinant vector of any one of claims 5 to 8. AMENDED SHiEET I- U ZZ:41 UHMNIMAU VALIUM AUMIL5 I I- I-j t -I 0. j 'I I- h UZI P.lv/zl iraviou 25 A therapeutic composi4,tion comprising a therapeutically effective amount of a lysostaphin analog as set forth in any one of claims 1 to 3 in association with a pharmaceutically acceptable carrier.
11. A use of lysostaphin analog as set forth in any one of claims 1 to 3 for making a medication for treating a disease caused by Staphylococcus species.
12. The use as set forth in claim 11, wherein the disease is mastitis.
13. A method of producing a lysostaphin analog which comprises the steps of; is a) transfecting an eukaryotic cell cr tissue with an expression vector as defined in any one of claims 5. to 7; b) allowing the expression of said nucleic so as to produce che lysostaphin analog; and c) recovering the lysostaphin analog.
14. A method o.-f producing a lysostaphin analog which comprises the steps of: a) transfecting an eukaryotic cell or tissue with an expressionl vector as defined in claim 8; ID) allcw"In -the e.-pre-04-n of sald nucl.eic so as to produce the lysostaphin analog; and c) recovering the lysostaphin analog. The methods of c laim 13 or 1.4, eukaryotic cell or tioaue is mnammalian. wherein the
16. The method of claim 13 or 14, wherein the lysoetaphin analog is produced in -vitro. AMENDED SHEET .LiII-I*.ZZ-:41 UOa-MAHrInhAu RAW ACK i iE'S *j.jI' ti-[U dU 7 -26
17. The method of Claim 14, wherein the lysostaphin analog i~s produced in vivo.
18. A method of producing a lysostaphin analog in the milk of a ruminant mammal, said method comprising the steps of; a) administering an effective amount of a recombinant vector as set forth in claim 8 into a cistern, ductal tree, and/or alveoli of a mammary gland of a ruminant mammal; b) allowing the expression of said nuclei,.c acid and secretion of the lysostapbhin analog into the milk of said animal; c) collecting milk from said mammal; and is d) purifying the lysostaphin analog from the collected milk.
19. The method of claim 18, wherein said mammal is a bovine, a sheep, a goat or a porcine. The method of claim l8 or 19 wherein said vector is treated to enhance its ability to cross the membrane of an epithelial cell.
21. The method of claim 20, wherein the treatment of said vector comprises forming a complex with a polycasicnic compound.
22. The method of claim 21, wherein said polycatior-ic compound is selected from the group consisting cof Poly- lysine and poly-ornitine.
23. The method of claim 20, wherein the treatment of said vector comprises forming a complex with a lip-Ld. ANIFNDE i -'E j .4!3 MU: Us-MAXI INkAU WALKER AUt.IE fliI4-'1f4-4 ug rav US -27
24. The method of claim 23, wherein said lipid, is cationic. The method of claim 20, wherein the treatment of said vector~ comprises formning a complex with a polyamine.
26. The method of claim 20, wherein the treatment of said vector comprises forming a complex with an amphiphillic: compound.
27. The method of any one of claims 20 to 25, where-i the treatment of said vector includes cholesterol. 2a8. The mar-had of any one of claim IS to 27, wherein said recombinant vector is administered by infusion of said vector in a liquid carrier into a target tissue.
29. A compositiun comprising a recombinant vector as set forth in any one of claims 5 to 7 anrd an acceptabl.e carrier. A composition comprising a recombinant vector as met forth in claim a and an acceptable carrier. 31, A comosition as set forth in claim 30, wherein said carrier is a pharmaceuti~call.y accept~able carrier. A 1 q E[E Sr-"!E
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2211293 CA2211293A1 (en) | 1997-07-23 | 1997-07-23 | Recombinant lysostaphin analogs |
| CA2211293 | 1997-07-23 | ||
| PCT/CA1998/000679 WO1999005289A1 (en) | 1997-07-23 | 1998-07-10 | Recombinant lysostaphin analogs |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU8328598A AU8328598A (en) | 1999-02-16 |
| AU734683B2 true AU734683B2 (en) | 2001-06-21 |
Family
ID=4161105
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU83285/98A Ceased AU734683B2 (en) | 1997-07-23 | 1998-07-10 | Recombinant lysostaphin analogs |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0998570A1 (en) |
| AU (1) | AU734683B2 (en) |
| CA (1) | CA2211293A1 (en) |
| NZ (1) | NZ502451A (en) |
| WO (1) | WO1999005289A1 (en) |
Families Citing this family (8)
| 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 |
| US20030211995A1 (en) * | 2001-12-21 | 2003-11-13 | Kokai-Kun John Fitzgerald | Methods and formulations for eradicating or alleviating staphylococcal nasal colonization using lysostaphin |
| AU2002367820B2 (en) * | 2001-12-21 | 2007-11-01 | Biosynexus Incorporated | Truncated lysostaphin molecule with enhanced staphylolytic activity |
| 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 |
| CN102533821B (en) * | 2011-12-12 | 2013-06-05 | 西北农林科技大学 | Recombinant lysostaphin gene and expression vector and recombinant cell constructed by recombinant lysostaphin gene |
| WO2015175774A1 (en) | 2014-05-14 | 2015-11-19 | Trustees Of Dartmouth College | Deimmunized lysostaphin and methods of use |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9415167D0 (en) * | 1994-07-27 | 1994-09-14 | Springer Caroline J | Improvements relating to cancer therapy |
| GB9509461D0 (en) * | 1995-05-10 | 1995-07-05 | Immunova | DNA molecules and constructs and their use in the treatment of mastitis |
-
1997
- 1997-07-23 CA CA 2211293 patent/CA2211293A1/en not_active Abandoned
-
1998
- 1998-07-10 NZ NZ502451A patent/NZ502451A/en unknown
- 1998-07-10 AU AU83285/98A patent/AU734683B2/en not_active Ceased
- 1998-07-10 EP EP98933404A patent/EP0998570A1/en not_active Withdrawn
- 1998-07-10 WO PCT/CA1998/000679 patent/WO1999005289A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| CA2211293A1 (en) | 1999-01-23 |
| AU8328598A (en) | 1999-02-16 |
| NZ502451A (en) | 2002-02-01 |
| WO1999005289A1 (en) | 1999-02-04 |
| EP0998570A1 (en) | 2000-05-10 |
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