WO1996040938A1 - Production de chondroitinase dans les souches de proteus vulgaris recombinees - Google Patents

Production de chondroitinase dans les souches de proteus vulgaris recombinees Download PDF

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WO1996040938A1
WO1996040938A1 PCT/US1996/008509 US9608509W WO9640938A1 WO 1996040938 A1 WO1996040938 A1 WO 1996040938A1 US 9608509 W US9608509 W US 9608509W WO 9640938 A1 WO9640938 A1 WO 9640938A1
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chondroitinase
plasmid
vulgaris
cell
plp2
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PCT/US1996/008509
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English (en)
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Jason Arnold Lotvin
Kiran Manohar Khandke
Mark Edward Ruppen
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American Cyanamid Company
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Priority to JP9501087A priority Critical patent/JPH11507512A/ja
Priority to EP96917959A priority patent/EP0833926A1/fr
Priority to AU60334/96A priority patent/AU6033496A/en
Publication of WO1996040938A1 publication Critical patent/WO1996040938A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

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  • This invention relates to the preparation, identification, and isolation of a plasmid that encodes for the production of large amounts of chondroitinase I and chondroitinase II proteins in Proteus vulgaris cells in the absence of natural exogenous chondroitinases I and II inducers such as chondroitin sulfate.
  • Chondroitinases are enzymes of bacterial origin that act on chondroitin sulfate, a component of the proteoglycans that mediate the attachment between the retina and the vitreous body of the human eye.
  • chondroitinase enzymes are chondroitinase ABC, which is produced by the bacterium Proteus vulgaris (P. vulgaris) , and chondroitinase AC, which is produced by A. aurescens .
  • Chondroitinases ABC and AC function by degrading polysaccharide side chains in protein-polysaccharide complexes, without degrading the protein core.
  • Yamagata et al. J. Biol . Chem. l:1523-1535, 1968 describe the purification of the chondroitinase ABC from extracts of P. vulgaris .
  • This enzyme selectively degrades the glycosaminoglycans chondroitin-4-sulfate, dermatan sulfate, and chondroitin-6-sulfate (also referred to respectively as chondroitin sulfates A, B, and C which are side chains of proteoglycans) at pH 8 at higher rates than it degrades chondroitin or hyaluronic acid.
  • the products of the degradation are high molecular weight unsaturated oligosaccharides and an unsaturated disaccharide.
  • chondroitinase ABC does not act on keratosulfate, heparin or heparitin sulfate.
  • chondroitinases include rapid, specific and non-surgical disruption of the attachment of the vitreous body to the neural retina of the eye, thereby facilitating removal of the vitreous body. See, for example, Hageman, U.S. Patent No. 5,292,509.
  • Chondroitinase ABC is designated as chondroitinase I in the present invention.
  • P. vulgaris chondroitinase I migrates with an apparent molecular mass of about 110 kDa when resolved by SDS-PAGE.
  • the appearance of a doublet in SDS-PAGE resolution of chondroitinase I has been reported (Sato et al. , Agric. Biol . Chem. ⁇ 0.:4,1057- 1059, 1986) .
  • this doublet represents intact chondroitinase I and a 90 kDa degradation product (U.S. Patent Application Nos.
  • Chondroitinase II is a polypeptide of 990 amino acids with an apparent molecular mass by SDS-PAGE of about 112 kDa. Its molecular mass as determined by electrospray and laser desorption mass spectrometry is 111,772 ⁇ 27 and 111,725 ⁇ 20 daltons, respectively. Chondroitinase II has an isoelectric point of 8.4-8.45. Its enzymatic activity is distinct from, but complementary to, that of chondroitinase I .
  • Chondroitinase I endolytically cleaves proteoglycans to produce end-product disaccharides, as well as at least two other products which are thought to be tetrasaccharides. Chondroitinase II digests at least one of these tetrasaccharide products of chondroitinase I digestion of proteoglycan.
  • Native or wild-type P. vulgaris bacterial strains typically do not produce significant amounts of chondroitinases I and II under ordinary growth conditions.
  • Wild-type strains of P. vulgaris can be induced to produce detectable levels of chondroitinase by providing an inducing substrate, such as chondroitin sulfate, as the sole carbon source.
  • chondroitin sulfate which is obtained from shark cartilage, is expensive and available only in limited quantities.
  • cloned chondroitinase I and II genes in E. coli can be expressed using a heterologous expression system with an artificial inducer, which also increases the cost of chondroitinase I and II production.
  • Plasmids pLP 2 -1531, which encodes chondroitinases I and II, and pLP 2 -1521, which encodes chondroitinase I are provided. These plasmids are used to transform wild-type E. coli and P. vulgaris cells. The transformed cells produce DNA encoding the production of chondroitinases I and II or chondroitinase I in the absence of natural exogenous chondroitinases I and II inducers. Additionally, the transformed P. vulgaris cells, in the absence of exogenous chondroitinases I and II inducers, produce the respective enzymes in amounts in excess of those produced by wild-type P. vulgaris in the presence of exogenous chondroitinases I and II inducer(s) .
  • Starting plasmid pLP 2 -751 and intermediate plasmids pLP 2 -770, pLP 2 -1512, pLP 2 -1263, pLP -1508, pLP 2 - 1510, pLP 2 -1514, pLP 2 -1518, and pLP 2 -1525 are also provided. These intermediate plasmids are typically used in the preparation of pLP 2 -1531 and pLP 2 -1521.
  • a Bglll/EcoRI fragment of about 3970 bp derived from pLP 2 -1512, a EcoRI/Smal fragment of about 2550 bp derived from pLP 2 -1514, and a Bglll/Smal (Aval) fragment of about 6520 bp derived from pLP 2 -1518 are further disclosed.
  • chondroitinases I and II are recovered by
  • Figure 1 is a schematic illustration of the preparation of pLP 2 -1518 from pLP 2 -751.
  • FIG. 2 is a schematic illustration of the preparation of pLP 2 1531 from pACYC184 and pLP 2 -1518. Detailed Description of the Invention
  • the present invention encompasses constructs and methods for producing large quantities of chondroitinase I and II proteins in Proteus vulgaris .
  • Chondroitinase I and chondroitinase II are enzymes produced by P. vulgaris that catalyze the breakdown of chondroitin sulfate, including that present in proteoglycans.
  • the physical and enzymatic characteristics of each enzyme are summarized in Table 1.
  • Wild-type strains of P. vulgaris accumulate easily detectable levels of enzymatically active chondroitinases I and II only when grown in a culture containing an exogenous chondroitinase I and II inducer, such as chondroitin sulfate, as the sole carbon source.
  • an exogenous chondroitinase I and II inducer such as chondroitin sulfate
  • Growth of wild-type strains of P. vulgaris in media without such an inducer such as, for example, in a rich medium containing many carbon sources or in a minimal medium containing, for example, glucose as a sole carbon source, results in insignificant or no detectable accumulation of chondroitinase I or II activity.
  • DNA encoding P. vulgaris chondroitinase I and/or chondroitinase II protein(s) is cloned into a plasmid that replicates in P. vulgaris cells.
  • the plasmid is engineered so that the chondroitinase I and II genes are placed immediately 3' to a sequence that serves as a constitutive promoter.
  • a promoter is a DNA sequence that stimulates transcription of the gene sequences that are located immediately downstream of, i.e., 3' to, the promoter sequence.
  • a constitutive promoter is a DNA sequence that stimulates transcription of downstream sequences (for example, chondroitinase I and/or II-encoding sequences) independent of the presence of exogenous transcriptional inducers.
  • the promoters or constitutive promoters of the present invention may be derived from the native chondroitinase I promoter typically present upstream of the chondroitinase I gene in wild-type P. vulgaris cells, if the promoter sequence is engineered so that regulatory sequences involved in induction of expression by exogenous inducers, such as, for example, chondroitin sulfate, are absent or are not functional.
  • the promoter to which the chondroitinase I and II genes are linked may comprise another known constitutive promoter that stimulates transcription in P. vulgaris, such as the promoter region of other P. vulgaris genes, or the promoter region of E.
  • coli genes that, for example, confer resistance to antibiotics such as, for example, chloramphenicol, tetracycline, and ampiciUin in P. vulgaris.
  • antibiotics such as, for example, chloramphenicol, tetracycline, and ampiciUin in P. vulgaris.
  • sequence of both the homologous chondroitinase promoter or that of heterologous promoters may be altered by deletions, insertions, and substitutions, using recombinant DNA methods that are well-known in the art. (See, for example, Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1982).
  • plasmids suitable for use in the present invention include sequences that allow the plasmid to replicate to high copy number in P. vulgaris, as well as sequences that encode a selectable marker.
  • Selectable markers may comprise gene products that confer resistance to tetracycline , ampiciUin, chloramphenicol, kanamycin, or streptomycin.
  • the chondroitinase I and/or H-encoding plasmid includes a gene encoding a chloramphenicol-resistance trait, such as, for example, chloramphenicol acetyl transferase.
  • suitable plasmids include without limitation pBR322, pNEB193, pUC19, and derivatives therefrom. (New England Biolabs, Beverly, MA).
  • a plasmid encoding chondroitinase I and/or chondroitinase II protein under the control of a constitutive promoter is introduced into P. vulgaris cells.
  • Suitable host cells include any strain of P. vulgaris, including without limitation wild- type P. vulgaris (for example, ATCC strain 6896), mutant P. vulgaris strains such as those described in U.S. Patent Application No. 08/476,261 filed June 7, 1995 (attorney docket # 0646/0B123).
  • Introduction of the plasmid may be achieved by any DNA transformation method well-known in the art, such as, for example, electroporation or calcium-mediated permeabilization of cells.
  • electroporation is used to achieve effective uptake of DNA into P. vulgaris host cells.
  • Selection of useful and preferred chondroitinase I and/or II-encoding plasmids is achieved by transforming individual P. vulgaris cultures with each plasmid, selecting transformants, and analyzing the transformants for their production of chondroitinase I and/or chondroitinase II.
  • colony screening methods can be used for preliminary assessment of relative chondroitinase production (as when, for example, a nested set of promoter deletions is analyzed).
  • the native chondroitinase I upstream region (including the presumptive promoter) is subjected to exonucleolytic cleavage with, for example, Bal 31 to generate a nested set of 5' deletion mutants.
  • the mixture of mutants is used to transform P. vulgaris.
  • the library of clones is spread and subjected to screening for chondroitinase production in the absence of an exogenous inducer.
  • Suitable screening methods include, but are not limited to, those that detect colonies that either bind chondroitinase-specific antibodies or that catalyze the breakdown of chondroitin sulfate or proteoglycan.
  • Colony immunoblotting assays use well-known methods such as those disclosed in, for example, Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory (1988).
  • Detection assays that depend on chondroitinase enzymatic activity include, without limitation, the chondroitin depletion method described below and colorimetric assays that detect chondroitin or chondroitin hydrolysis. Either screening method (immunoblotting and chondroitin depletion) can be used at any of the stages in initial identification and subsequent colony purification of constitutive mutant cells.
  • an antibody-based screening method is used to identify P. vulgaris colonies that produce chondroitinases I and II even when an exogenous inducer such as, for example, chondroitin sulfate is absent from the culture medium as follows:
  • P. vulgaris cultures are seeded onto filters that are placed onto agar plates containing either rich or casamino acid-supplemented minimal medium lacking chondroitin sulfate and/or N-acetylgalactosamine.
  • the filters may comprise nylon, paper, nitrocellulose, or polyvinylidiene difiuoride, preferably NYTRAN (Schleicher & Schuell, Keene, NH).
  • NYTRAN Schlescher & Schuell, Keene, NH
  • the optimum colony density for this step is about 1000 per plate.
  • the filters are then transferred from the plates to a solution that permeabilizes and lyses the cells on the filter, so that chondroitinase polypeptides are released from the cells and become fixed to the filter.
  • Goat anti-chondroitinase I antibodies are prepared by (a) purifying P. vulgaris chondroitinase I from an E. coli strain that expresses chondroitinase I from an overexpression plasmid (as disclosed in U.S. patent application No. 08/233,008, filed April 22, 1994 hereinafter the '008 application) using purification methods described in U.S. patent application No. 08/231,534, filed April 22, 1994; and (b) mixing the purified chondroitinase I with Freund's adjuvant and inoculating the resulting emulsion into goats.
  • Rabbit anti-chondroitinase II antibodies are prepared by (a) purifying P. vulgaris chondroitinase II from an E.
  • coli strain that expresses chondroitinase II from an over expressing plasmid (as disclosed in the '008 application) and (b) mixing the purified chondroitinase II with Freund's adjuvant and inoculating the resulting emulsion into rabbits.
  • Procedures for the purification and analysis of antibodies are those well-known in the art.
  • P. vulgaris colonies are grown on Nytran filters that are placed onto agar plates containing "20-10-5" medium, which includes 20 g/1 tryptone, 10 g/1 yeast extract, and 5 g/1 NaCl. After spraying the bacterial colony-containing filters with a solution of bovine serum albumin to block background sites, the filters are floated on liquid chloroform for several hours or placed on chloroform-saturated paper, releasing chondroitinases and binding them to the filter in the immediate vicinity of chondroitinase-producing colonies.
  • the washed filters are then incubated sequentially with (1) goat anti- chondroitinase I antibodies, (2) peroxidase-conjugated rabbit anti-goat antibody (BioRad), and (3) color reagents to visualize filter-bound peroxidase (BioRad).
  • colony-purified untransformed wild-type P. vulgaris cells are grown on medium lacking and containing chondroitin sulfate to serve as negative and positive controls, respectively, for the screening procedure. Colonies that display detectable amounts of chondroitinase I and/or II using this assay are picked, diluted, re-inoculated on plates, and the entire detection procedure is repeated.
  • chondroitin depletion assay is used to identify chondroitinase I and/or ⁇ -producing colonies.
  • mutagenized P. vulgaris cultures are seeded onto filters as described above, and the filters are placed onto agar plates containing either rich or casamino acid-supplemented minimal medium lacking chondroitin sulfate.
  • the filters are transferred to plates containing agar supplemented with 5 mg of chondroitin sulfate/ml and a protein synthesis inhibitor at a concentration effective to inhibit protein synthesis on the filter-bound bacterial colonies, preferably 100 ⁇ g/ml tetracycline.
  • a protein synthesis inhibitor at a concentration effective to inhibit protein synthesis on the filter-bound bacterial colonies, preferably 100 ⁇ g/ml tetracycline.
  • the filters are removed, and the plates are flooded with about 10 ml each of 0.5% cetyl pyridinium chloride (Sigma Chemical Co., St. Louis, MO). This treatment causes chondroitin sulfate to form a cloudy precipitate within the agar. Colonies that elaborate chondroitinase I and II produce an obvious clear zone surrounding the colony that is easily visualized by eye.
  • Chondroitinase-encoding plasmids that appear by the above screening assays to provide detectable production of chondroitinases I and II in the absence of the exogenous inducers chondroitin sulfate or N-acetylgalactosamine are analyzed for chondroitinase production in a quantititative manner. Individual colonies are inoculated into small-scale (3-5 ml) fermentation cultures and incubated at 30-37 °C with shaking. Samples are removed at different times after initiation of growth, cells are disrupted using a French pressure cell, and the amount of chondroitinase protein and enzymatic activity is measured in the cell homogenates.
  • Useful plasmids are those that result in the production of at least 0.2 chondroitinase activity units per A ⁇ o unit of bacterial culture.
  • expression of chondroitinase I and/or chondroitinase II using the plasmids of the present invention results in the production of at least 0.5 chondroitinase activity units per A m unit of bacterial culture.
  • the chondroitinase I and II enzymes produced by a transformed P. vulgaris strain may he co-purified to homogeneity (i.e., to obtain a pure mixture of chondroitinase I and II) and the co-purified enzymes are suitable for use in, for example, vitreal disinsertion.
  • a preferred affinity chromatography includes:
  • the proteins can be further purified by metal chelating chromatography by (1) contacting the unbound eluate with a metal chelating affinity chromatography support to bind further the chondroitinase proteins;
  • the copurified proteins can be separated from each other by additional process steps involving further cation exchange chromatography.
  • the individually purified proteins can be used in ratios other than those obtained by the copurification procedure.
  • the overall strategy was to create a DNA fragment containing the chondroitinase I and II genes but lacking most of the upstream regulatory region. It was desired that the final fragment be flanked by Bglll-Smal sites so that it could be inserted into a plasmid vector containing appropriately placed Bglll and Smal sites.
  • the chondroitinase region was initially engineered in halves and then was reassembled to achieve this result.
  • chondroitinase I-encoding DNA is designated “110 K” and chondroitinase II-encoding DNA is designated “112 K”.
  • the starting plasmid was a cosmid clone designated pLP 2 -751, which contains a ⁇ 30kb P. vulgaris genomic DNA insert flanked by EcoRI sites. Since the insert contains an internal EcoRI site, EcoRI digestion results in recovery of the inserted DNA as two EcoRI fragments of 20kb and lOkb, which are arbitrarily designated as left and right, respectively.
  • the entire chondroitinase I gene is located on the extreme right side of the 20kb DNA fragment.
  • the chondroitinase II gene is located immediately downstream to the right of the chondroitinase I gene.
  • the proximal portion of this gene is at the rightmost end of the 20kb EcoRI fragment, while the remaining, distal, portion of the gene is on the lOkb EcoRI fragment.
  • the coding sequence for the chondroitinase II gene extends rightward and terminates about
  • pLP 2 -751 also contains a lOkb Nsil fragment that contains the entire chondroitinase I gene and the proximal portion of the chondroitinase II gene. This fragment maps to the right side of the pLP 2 -751 20kb EcoRI fragment, but extends
  • the lOkb Nsil fragment contains the internal EcoRI site described above.
  • pLP 2 -751 The overlapping lOkb Nsil and lOkb EcoRI fragments from pLP 2 -751 were subcloned as follows. pLP 2 -751 was digested with Nsil, and the lOkb Nsil fragment was isolated and cloned into a pEBI24 derivative (International Biotechnologies, Inc., New Haven, CT) containing an Nsil linker. This step yielded a plasmid designated LP 2 -770 ( Figure 1).
  • pLP 2 -770 was then digested with EcoRV to remove about 3700 bp of unsequenced upstream DNA that presumably contains the regulatory gene for the chondroitinase operon and possibly all or part of the regulatory binding site for theses proteins at the 5 '-end of the chondroitinase I gene.
  • the EcoRV ends were dephosphorylated, after which Bglll linkers were ligated to the dephosphorylated ends.
  • the resulting plasmid was designated LP 2 -1512. Immediately downstream of the Bglll site are the presumed chondroitinase promoter -35 and -10 elements beginning at nucleotides 40 and 62.
  • the start codon for chondroitinase I is at nucleotide 119.
  • the chondroitinase I termination codon is at nucleotide 3182, and the start codon for chondroitinase II is at position 3238.
  • the DNA contains an EcoRI site at position 3974, corresponding to that contained in plasmid LP 2 -1514 (see below), and extends to the Nsil site at position 4363. Accordingly, digestion of pLP 2 - 1512 with Bglll and EcoRI produced an approximately 3970 bp fragment containing the entire chondroitinase I gene and the 5' proximal portion of the chondroitinase II gene.
  • plasmid LP 2 -751 was digested with EcoRI, and the 10 kb EcoRI fragment described above was cloned into the EcoRI site of a pNEB193 derivative (New England Biolabs, Beverly, MA) to yield a plasmid designated LP 2 -
  • plasmid LP 2 - 1510 This step removed about 7450 bp of DNA downstream of the chondroitinase II coding sequence.
  • pLP 2 -1510 was then digested at a sole vector-derived Aaffl site, filled in with the Klenow fragment of DNA polymerase I, dephosphorylated, and a Bglll linker was ligated at that site.
  • the resulting plasmid, designated LP 2 -1514 contains an approximately 2550 bp EcoRI/SmaI(AvaI) insert containing the 3' distal portion of the chondroitinase II gene.
  • the resulting plasmid designated LP 2 -1518, has an approximately
  • Plasmid pACYC184 (ATCC #37033) was engineered to serve as a receptor for the BglH-Smal (Aval) chondroitinase fragment described above. The steps of this construction are shown in Figure 2.
  • pACYC184 was digested with Aval, after which the ends were filled in using the Klenow fragment of DNA polymerase, dephosphorylated, and ligated to a synthetic Bgl ⁇ linker to form plasmid LP 2 -1508. This plasmid was then digested with Hindi ⁇ , filled in, dephosphorylated, and ligated to synthetic Smal linkers to form plasmid LP 2 -1525.
  • pLP -1525 was digested with Bglll and Aval and was dephosphorylated.
  • the approximately 2850 bp fragment (including the origin of replication and chloramphenicol resistance gene) was then ligated to the Bglll/ A val insert containing the chondroitinase I and II fragment to yield plasmid LP -1531.
  • E. coli strain carrying this plasmid is designated LL4136.
  • a chondroitinase I only construct was also prepared by inserting a Bglll to SphI (position 3414) fragment from pLP 2 -1512 into a Bglll-SphI digested derivative of pACYC184 which contains a Bglll linker at the ⁇ ACYC184 Bell site; the SphI site is internal to the pACYC184 tetracycline-resistance gene.
  • the resulting plasmid is designated LP 2 -1521 and is contained in E. coli strain LL4093.
  • Proteus vulgaris strains LL2480 (wild-type) and LL2492 (exhibiting constitutive expression of chondroitinases) were transformed with plasmids using the following procedure.
  • the electroporated cells were then inoculated into 2ml of 20-10-5 medium, and the cultures were incubated at 37 °C for about 75 min, after which they were plated on 20-10-5 agar containing 25 ⁇ g/ml chloramphenicol. After overnight incubation at 37 °C, chloramphenicol-resistant colonies were observed. At least one colony of each transformant was streaked onto chloramphenicol agar. Individual colonies were then inoculated into 20-10-5 liquid medium containing 25 ⁇ g/ml chloramphenicol and grown overnight at 37 °C. Glycerol was then added and the strains were stored at -70 °C.
  • the plasmids transformed were pACYC184, pLP 2 -1521 and pLP 2 -1531.
  • Strain LL2480 transformed with plasmids p AC YC 184, pLP " 1521 andpLP 2 -1531 were designated strains LL4202, LL4198 and LL4192, respectively.
  • Strain LL2492 transformed with pACYC184, pLP 2 -1521, and pLP 2 -1531 were designated strains LL4119, LL4107, and LL4142, respectively.
  • Example 3 Analysis of Chondroitinases I and II Production
  • the plasmid-transformed P. vulgaris strains prepared as described in Example 2 above were analyzed for their production of chondroitinases I and II.
  • the growth medium was casamino acid-supplemented minimal medium containing 25 ⁇ g/ml chloramphenicol. Incubations were performed at 30 °C and samples were taken at 7 and 24 hours after initiation of growth. The starting cell densitites were about 10 6 -10 7 cells/ml. In each case, cells were collected by centrifugation, disrupted in a French pressure cell, and subjected to chondroitinase enzymatic activity assays. The results, expressed as activity units/ Agoo units of the culture, are shown in Table 2.
  • pLP 2 -1531 confers increased production of chondroitinases I and II in both constitutive and regulated Proteus host strains.
  • Plasmid LP 2 -1521 confers increased production of chondroitinase I only.
  • a regulated strain background high levels of chondroitinase I are seen, but no chondroitinase II is observed.
  • pLP 2 -1521 confers high levels of chondroitinase I and low levels of chondroitinase II. The production of chondroitinase II in this strain is attributable to expression from the chromosomal copy of this gene.

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Abstract

L'invention se rapporte au plasmide pLP2-1531, qui code les chondroitinases I et II, et au plasmide pLP2-1521, qui code la chondroitinase I. On utilise ces plasmides pour transformer les cellules E. coli et P. vulgaris de type sauvage. Les cellules transformées produisent de l'ADN qui code la production des chondroitinases I et II ou de la chondroitinase I en l'absence des inducteurs exogènes naturels des chondroitinases I et II. En outre, les cellules transformées de P. vulgaris produisent en l'absence des inducteurs exogènes des chondroitinases I et II les enzymes respectifs dans des proportions supérieures à celles produites par le P. vulgaris de type sauvage en présence du ou des inducteurs exogènes des chondroitinases I et II. Le plasmide de départ pLP2-751 et les plasmides intermédiaires pLP2-770, pLP2-1512, pLP2-1263, pLP2-1508, pLP21510, pLP2-1514, pLP2-1518, et pLP2-1525 sont également décrits dans l'invention. Ces plasmides intermédiaires sont généralement utilisés dans la préparation des plasmides pLP2-1531 et pLP2-1521. On décrit par ailleurs un fragment BglII/EcoRI d'environ 3970 bp provenant du plasmide pLP2-1512, un fragment EcoRI/SmaI d'environ 2550 bp provenant du plasmide pLP2-1514, et un fragment BglII/SmaI(AvaI) d'environ 6520 bp provenant du plasmide pLP2-1518. Enfin, on envisage l'utilisation des cellules transformées de P. vulgaris en vue de produire la chondroitinase I et/ou la chondroitinase II. Lesdites cellules P. vulgaris sont mises en culture dans un milieu de culture bactérienne, en l'absence d'inducteur exogènes des chondroitinases I et II. Une fois les cellules recueillies, on peut extraire les enzymes que constitue la chondroitinase I et/ou la chondroitinase II.
PCT/US1996/008509 1995-06-07 1996-06-04 Production de chondroitinase dans les souches de proteus vulgaris recombinees WO1996040938A1 (fr)

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JP9501087A JPH11507512A (ja) 1995-06-07 1996-06-04 組み換えプロテウス ブルガリス（Ｐｒｏｔｅｕｓ ｖｕｌｇａｒｉｓ）株におけるコンドロイチナーゼの産生
EP96917959A EP0833926A1 (fr) 1995-06-07 1996-06-04 Production de chondroitinase dans les souches de proteus vulgaris recombinees
AU60334/96A AU6033496A (en) 1995-06-07 1996-06-04 Chondroitinase production in recombinant proteus vulgaris st rains

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9796970B1 (en) 2017-04-24 2017-10-24 Advantek Serum Laboratories Ltd. Production of high purity chondroitinase ABC

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0613949A2 (fr) * 1993-02-24 1994-09-07 Maruha Corporation Gène codant pour la chondroitinase ABC et usages de celui-ci
WO1994025567A1 (fr) * 1993-04-23 1994-11-10 American Cyanamid Company CLONAGE ET EXPRESSION DE GENES DE CHONDROITINASE I ET II A PARTIR DE $i(P. VULGARIS)

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EP0613949A2 (fr) * 1993-02-24 1994-09-07 Maruha Corporation Gène codant pour la chondroitinase ABC et usages de celui-ci
WO1994025567A1 (fr) * 1993-04-23 1994-11-10 American Cyanamid Company CLONAGE ET EXPRESSION DE GENES DE CHONDROITINASE I ET II A PARTIR DE $i(P. VULGARIS)

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US9796970B1 (en) 2017-04-24 2017-10-24 Advantek Serum Laboratories Ltd. Production of high purity chondroitinase ABC

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CA2223980A1 (fr) 1996-12-19
AU6033496A (en) 1996-12-30
EP0833926A1 (fr) 1998-04-08
JPH11507512A (ja) 1999-07-06

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