WO1994000580A1 - CLONAGE MOLECULAIRE DES GENES RESPONSABLES DE LA PRODUCTION DE COLLAGENASE A PARTIR DE $i(CLOSTRIDIUM HISTOLYTICUM) - Google Patents

CLONAGE MOLECULAIRE DES GENES RESPONSABLES DE LA PRODUCTION DE COLLAGENASE A PARTIR DE $i(CLOSTRIDIUM HISTOLYTICUM) Download PDF

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WO1994000580A1
WO1994000580A1 PCT/US1993/005944 US9305944W WO9400580A1 WO 1994000580 A1 WO1994000580 A1 WO 1994000580A1 US 9305944 W US9305944 W US 9305944W WO 9400580 A1 WO9400580 A1 WO 9400580A1
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collagenase
histolyticum
lys
gly
asp
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PCT/US1993/005944
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English (en)
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Hun-Chi Lin
Shau-Ping Lei
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Trigen, Inc.
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Priority to EP93916670A priority Critical patent/EP0654084A4/fr
Priority to JP6502521A priority patent/JPH08500970A/ja
Priority to AU46447/93A priority patent/AU4644793A/en
Publication of WO1994000580A1 publication Critical patent/WO1994000580A1/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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to the isolation and cloning of genetic information coding for Clostridium histolyticum collagenase and the expression of the genetic information in a suitable host.
  • this invention is directed to the isolation and cloining of genetic information coding for forms of Clostridium histolyticum collagenase, including a form having a molecular weight higher than the products of translation determined by the native expression of the C. histolyticum genomic coding sequence.
  • a recombinant DNA segment which codes for a polypeptide having the enzymatic activity and antigenicity of Clostridium histolvticiu collagenase.
  • This polypeptide is distinguishable from native C. histolyticum collagenases, that is, collagenases produced by the native expression of the C. histolyticum genome and subsequently purified from C. histolyticum.
  • This polypeptide of the invention referred to herein as a non- native form, has a higher molecular weight than the products of translation determined by the native expresion of the C. histolyticum genomic coding sequence.
  • the claimed recombinant DNA segment comprises a promoter derived from C. histolyticum. This promoter operates independently and allows the claimed DNA segment to be transcribed under the control of said promoter to produce the claimed polypeptide without the functioning of a promoter external to the claimed recombinant DNA segment.
  • the claimed recombinant DNA segment is further capable of expressing native polypeptides with collagenase activity having molecular weights lower than the claimed non-native, high molecular weight polypeptide.
  • the invention further provides a vector comprising the claimed recombinant DNA segment and capable of transforming host cells to produce the claimed non-native polypeptide.
  • the invention further provides other E. coli host cells transformed with the vector of the invention. These host cells produce a polypeptide possessing collagenase activity and antigencity and having a molecular weight of 110,000. Greater than 50% of the polypeptides having collagenase activity produced by these cells comprises the 110 kd collagenase.
  • Another aspect of the invention involves substantially purified preparations of C. histolyticum collagenase.
  • One preparation comprises the non-native form of collagenase.
  • Another substantially purified preparation of the invention comprises collagenase having a molecular weight of 110 kd. These forms of collagenase are derived from different strains of E. coli host cells transformed with the vector of the invention.
  • Further provides are methods for using the collagenase produced by the E. coli host cells genetically engineered according to the invention. These methods are suitale for such purposes as digesting connective tissue and releasing embedded cells, isolating dispersed pancreatic islets from pancreatic tissue, isolating endothelial cells from blood vessels, and dissociating tumors for isolation of dispersed tumor cells.
  • the method comprises two steps:
  • the step of separating the dispersed cells from tissue debris is typically performed by density gradient centrifugation.
  • the genetically engineered C. histolyticum collagenases of the present invention can also be used in a method for intradiscal treatment of herniation of nucleus pulposus ("slipped disc") .
  • This method comprises the steps of:
  • Figure 1 outlines the cloning strategy for obtaining expression plasmid for intact 125 kd collagenase.
  • Figure 2 shows the expression of collagenase in E_j_ coli strain DH5 ⁇ .
  • Figure 3 shows the construction of plasmids pCT6 and pCT7 for DNA sequencing of the collagenase gene.
  • Figure 4 shows various clones used to determine the partial DNA sequence of the gene encoding 125 kd collagenase.
  • Figure 5 is the transcriptional termination signal of the 125 kd collagenase gene.
  • Figure 6a is a Coomasie blue stained gel of 125 kd collagenase compared with commercially available collagenases
  • Figure 6b is a Western blot of 125 kd collagenase compared with commercially available collagenases.
  • Figure 7 shows in lane 1 a Coomasie blue stained gel of culture media from E. coli strain DH5 ⁇ containing plasmid p70; lane 2 is a Coomasie blue stained gel of purified 125 kd collagenase from culture media.
  • Figure 8 is the restriction of plasmid pCTll.10.
  • Figure 9 is a Coomasie blue stained SDS-PAGE showing that IPTG does not induce the expression of recombinant collagenase from E. coli DH5 ⁇ carrying pCTll.10.
  • Figure 10 is a Western immunoblot comparison of recombinant collagenase produced from pRS21 and pCT8B.
  • Figure 11a is a Coomasie blue stained SDS-PAGE; lib is an immunoblot, both showing the intracellular localization of the recombinant collagenase produced in E. coli.
  • Figure 12 shows the results of Coomasie blue staining and immunoblots of the purification and comparison of the recombinant 110 kd collagenase to natively produced 110 kd collagenase.
  • the inventors have cloned a gene for Clostridium histolyticum into E. coli.
  • the cloned gene that is, the recombinant DNA segment of the invention, is capable of expression as a polypeptide product in E. coli.
  • the product expressed in E. coli is detectable both in the form of protein immoreactive with anticollagenase antibody and in the form of assayable collagenase activity i.e. collagen digestion.
  • E. coli is detectable both in the form of protein immoreactive with anticollagenase antibody and in the form of assayable collagenase activity i.e. collagen digestion.
  • E. coli strain DH5 ⁇ transformed with the claimed recombinant DNA segment according to the invention, produced, in particular, a polypeptide having collagenase activity and antigenicity, the polypeptide being distinguishable from native C. histolyticum collagenases.
  • the distinguishing feature of this non-native polypeptide was that it had a higher molecular weight than the products of translation determined by the native expression of the C. histolyticum genomic coding sequence.
  • histolyticum collagenases determined by native expression are those forms of collagenase directly produced by C. histolyticum without the introduction of genetic vectors carrying DNA segments coding for collagenase.
  • native collagenases of C. histolyticum purchased from a variety of commercial sources do not have molecular weights as large as the non-native collagenase of the present invention.
  • the claimed recombinant DNA segment comprised a promoter derived from C. histolyticum.
  • the 4.9 kb recombinant DNA fragment of the invention coding for collagenase comprised a Clostridium promtoer which functioned independently in E. coli.
  • the claimed recombinant DNA segment was found to express polypeptides having collagenase activity and molecular weights ower than the non-native collagenase of the invention.
  • the relative proportions of native and non-native collagenases expressed from the claimed DNA segment vary according to the E. coli host into which the claimed DNA segment is transformed via the vector of the invention.
  • a pRK290 library containing clostridium DNA was constructed. (Example 1) and the collagenase gene screened.
  • the inserted collagenase gene was characterized by restriction enzyme analysis (Example 2) .
  • Example 4 Further characterization of the collagenase gene was carried out in Example 4, wherein the insert was placed in opposite insert orientations in plasmids pCT6 and pCT7, serially delted. 2551 base pairs were sequenced. A complete nucleotide sequence and inferred amino acid sequence is presented in Sequence ID No. 1. Further confirmation that the DNA sequence comprised the Clostridium collagenase gene is presented in Examples 5 and 6.
  • Clostridium collagenase genes in E. coli requires transformation or transfection of the host E____ coli cells with a suitable plamid or other vector carrying the Clostridium DNA and detection of the collagenase produced by the transformed cells.
  • a suitable plamid or other vector carrying the Clostridium DNA carrying the Clostridium DNA and detection of the collagenase produced by the transformed cells.
  • the inventors in Examples 3, 7, 9, 10 and 11 determined that the expresion of the claimed recombinant DNA segment in transformed host cells is determined by the strain of the host cell.
  • Example 3 demonstrates that about 2% of the transformed E. coli soluble proteins was collagenase according to immunoblotting with anti ⁇ collagenase antibodies on Western blots and Coomasie blue staining of SDS-PAGE.
  • Example 8 It was found, as described in Example 8, that control of collagenase expression in E. coli was under control of an independent promoter comprising the claimed DNA segment and derived from Clostridium histolyticum.
  • the inventors produced and purified native and non- native collagenase from transformed E. coli cells and from the culture medium in which these cells were grown, as described below in Example 10. Purification of the enzyme from cells involved standard technqieus known in the art. The inventors unexpectedly found large amounts of collagenase in the fermentation broth in which the transformed cells grew. Based on that observation, a strategy for purifying collagenase to obtain substantially pure preparations of non-native collagenase and 110 kd collagenase were developed.
  • the collagen digestion activity of recombinant collagenase purifed from transformed cells was found to be about equivalent to the activity of recombinant collagenase activity purified from cells. (Table 1 in Example 10) .
  • the collagen digestion activity of the purified recombinant non-native collagenase was compared to native forms of purified collagenases obtained commercially. (Table 2 in Example 11) the recombinant collagenase was 50% to 100% higher in activity than native collagenases obtained from Worthington Chemical, Sigma Chemical, and CalBiochem..
  • the purified recombinant non-native or recombinant native llOkd collagenase produced by genetically engineered E. coli containing the claimed DNA segment can be used for any application in which it is desired to digest collagen.
  • Particular applications for isolating or releasing cells from tissues include: (1) digesting connective tissue and releasing embedded cells without destroying cell membranes and other essential features; (2) isolating endothelial cells; (3) dissociating tumors; and (4) intradiscal treatment of herniation of the nucleus pulposus ("slipped disc").
  • the purified 110 kd recombinant collagenase of the invention, in combination with trypsin, was effective for isolating endothelial cells from human saphenous veins.
  • the amounts of recombinant collagenase (SEQUENCE ID NO. 2) , or native class II collagenase, or neutral proteases can be varied to obtain dissociated tissue preparations using the method of the present invention.
  • the type and amount of neutral proteases required in with the recombinant collagenase in the compositions and methods of the present invention can be varied depending on the target tissue.
  • Proteases other than crude or purifed native collagenases can be employed in compositions comprising the recombinant collagenase (SEQUENCE ID NO. 2) to digest or dissociate tissues using the method of the present invention.
  • the orientation of the gene encoding for the intact 125 kd collagenase was determined in U.S. Application Serial No. 07/498,919. As described below in Examples 4 and 5, the termination of the gene was determined by sequencing the gene and found to be close to the Bglll site. As defined by the above information, a 2.5 kb DNA fragment extended from the first EcoRI site through the second EcoRI site to the Bglll site. A 2.5 kb DNA fragment is not large enough to code for collagenase having a molecular weight of about 125 kd, which requires approximately 4.4 kb. Accordingly, a new library was prepared in an attempt to identify more collagenase gene sequence. This involved construction of a Bglll library.
  • the inventors employed another plasmid with a low copy number (1-10 copies per cell) , such as pRK290 (Haas, D. Experientia. 39:1199 (1983)). This resulted in the cloning of the entire collagenase gene. However, the difficulty of cloning the Bglll fragment to smaller plasmids, such as pUC8, remained. Nonetheless, the inventors achieved the construction of such a pUC8 plasmid incorporating the entire collagenase gene.
  • A. DNA isolation from Clostridium histolyticum Clostridium histolyticum ATCC 21000 was obtained from the American Type Culture Collection. The paper tablet containing the bacteria was first soaked in TYE broth (15 g tryptone, 10 g yeast extract, and 5 g NaCl per liter of culture medium) for 30 minutes at 4°C with occasional shaking.
  • the cell suspension was streaked on a TYE agar plate and grown at 37°C under anaerobic conditions. A single colony was picked and grown in 50 mL TYE broth and grown at 37 ⁇ C under anaerobic conditions. The cells were collected by centrifugation and resuspended in TES (0.1M Tris, pH8.0, O.lmM EDTA, and 0.15M NaCl) . Cells were partially lysed by freezing and thawing the cell suspension four times. SDS and pronase K were then added to final concentrations of 0.5% and 200 ⁇ g/mL, respectively.
  • TES 0.1M Tris, pH8.0, O.lmM EDTA, and 0.15M NaCl
  • the cell debris was removed by centrifugation at 10,000 rpm in a Beckman J2-21 centrifuge for 30 minutes at 4°C. The supernatant was extracted three times with equal volumes of phenol-chloroform, and the DNA was precipitated with isopropanol. DNA concentration was measured by electrophoresing the DNA through an agarose gel and comparing fluorescence after the addition of ethidium bromide with the fluorescence of a known concentration standard. The average size of purified Clostridium DNA was measured by electrophoresis on a 0.6% agarose gel and was 30 kb to 40 kb.
  • S&S dried nitrocellulose paper
  • the nitrocellulose paper was incubated with the rabbit anti-collagenase antibody (as detailed in Example 2 of U.S. Application Serial No. 07/498,919) at a dilution of 1:1000 with 5% skim milk at room temperature for 1 hour, washed with 0.1 M Tris-HCl buffer, pl ⁇ 8.0, containing 0.2 M NaCl and 1% TritonTM X-100, then incubated with protein A-horseradish peroxidase conjugate at 1:1000 dilution in PBS with 5% skim milk at room temperature for 1 hour. The washing procedure was repeated and the nitrocellulose paper developed with 4-chloro-l-naphthol and H 2 0 2 in PBS as described in R.A. Young & R.W. Davis, "Efficient Isolation of Genes by Using Antibody Probes," Proc. Natl. Acad. Sci. USA 80. 1194-1198 (1983).
  • DNA in plasmid P70 was subjected to further restriction enzyme analysis.
  • DNA from plasmid P70 was prepared as described (Maniatis) . Purified DNA was digested with Bglll to release the cloned DNA insert.
  • DNA fragment with apparent size of 4.9 kb was released from vector pRK290 by Bglll digestion.
  • p70 was further digested with a combination of Bglll and EcoRI and compared to pBBl and pRS21 digested with the same enzymes. All three plasmids released an identical 1.7kb EcoRI-Bglll fragment.
  • E. coli strain DH5 ⁇ was transformed with plasmids pUC8 or P70, respectively, and grown in 50 ml of TYE broth in 250 ml flask. The cells were harvested, resuspended in lOmM Tris, pH 7.4 and ImM PMSF, and sonicated. After removing the cell debri by centrifugation, protein concentrations were measured and the same amount of proteins were loaded and run on a 7.5% SDS polyacrylamide gel.
  • Panel A is Coomassie blue-stainned SDS-PAGE. Lane 1, E. coli strain DH5 ⁇ carrying plasmid pUC8 and Lane 2, E. coli strain DH5 ⁇ carrying plasmid P70.
  • Panel B is Coomassie blue-stainned (Lane 1) and immunoblotted (Lane 2) SDS-PAGE of cell extract prepared from E. coli strain carrying P70.
  • Plasmid P70 transformed E. coli strain DH5 ⁇ was grown in a 5 liter BioFloII Fermentor to produce collagenase. After cells were harvested by centrifugation, the cell paste was stored at -70°C. Small amount of cells were futher processed to determine the purification condition. Sonicated and clarified supernatant was examined by Coomassie blue stainning and immunoblotting with anti-collagenase antibodies and shown in the panel B of Figure 2.
  • the position of the Coomassie stainned band was the same position as the immunoreactive protein band. It therefore confirmed the identity of the Coomassie blue stainable band that resided between 200 and 97 kd was collagenase. Under both growth conditions, it was estimated that about 2% of E. coli soluble protein was collagenase.
  • Plasmids pCT6 and pCT7 were constructed by inserting the 2.5 kb BamHI-Bglll fragment from P42 to the BamHI site of plasmid pBluescript. Plasmids pCT6 and pCT7 represent opposite insert orientation as shown in Figure 3. To create unidirectional deletions, both plasmids were first digested with Xhol and KphI to create a recessed 3 1 - hydroxyl termini of double-stranded DNA and a protruding 3* termini at the other end.
  • the recessed 3'-hydroxyl termini created by Xhol was susceptable to exonuclease III and was be removed stepwise while the protruding 3' termini created by Kpnl remained intact.
  • the digestion proceeds unidirectionally away from the cleavage site and into the target DNA sequence. The degree of digestion was controlled by time. Aliquotes collected at different times were subjected to SI nuclease digestion. The digested DNA samples were analyzed by agarose gel electrophoresis to identify the samples containing DNA fragments with desired size. Klenow DNA polymerase was added to blunt both DNA ends. T4 DNA ligase was added to recircularize the plasmid. Ligated DNAs were then transformed into E. coli strain DH5 ⁇ .
  • the cells were plated on TYE-Ap plates, and grown overnight at 35°C.
  • the colonies were randomly picked and grown in 5 ml of TYE-Ap broth overnight. Cells were collected from overnight culture by centrifugation. The cell pellet was resuspended in Tri- EDTA buffer and lysed by using the alkaline method as described in Maniatis. After ethanol precipitation, the DNA pellet was briefly dried and resuspended in TE (lOmM Tris, pH 8.2, 0.1 mM EDTA). The size of deletion was examined by restriction enzyme analysis. Clones with appropriate deletions were identified and further treated with RNase A.
  • Plasmid pCT12.8 was constructed by inserting the 4.9 kb Bglll fragment of P70 into pBluescript (SK-) pretreated with BamHI.
  • Sequence ID No. 1 A total of 2808 base pairs of the complete sequence is shown in (Sequence ID No. 1) .
  • the DNA sequence contains an unusually high proportion of A and T nucleotides (69%) and only 31% of G and C nucleotides.
  • An open reading frame was identified from the first EcoRI site through the second EcoRI site and ends at a TAA termination codon located at base pair 2808.
  • a deduced protein sequence containing 936 amino acids was identified and shown in Sequence ID No. 2. This protein contains unusually high charged amino acids (30%) as compared to most other proteins.
  • a typical bacterial transcriptional termination signal (Platt, T. and D.G. Bear in Gene Function in Prokarvotes. J. Beckwith et al.
  • TAA translational termination signal
  • the DNA sequence flanked by multiple cloning sites was sequenced using the flanking universal primers. Flanking sequences of plasmids pBBl, pRS21, and P41 were determined and compared to the sequences obtained from subclones of P70. No differences was observed. This result suggested that the P70 shares the same DNA fragment as pBBl and was located at the same place on the chromosome.
  • coli strain DH5 ⁇ containing either plasmid pCT8B, pRS21, or pUC18 were grown in TYE-Ap broth overnight at 35° C. Cells were collected by centrifugation and disrupted with SDS-PAGE loading buffer. The released proteins were run on 7.5% SDS-polyacrylamide gel and electroblotted to nitrocellulose paper. The immunoreactive bands were detected as previously described and compared. As shown in lane 1 of Figure 10, lysate produced from ' E. coli carrying pUCl ⁇ did not produce an immunoreactive collagenase band. However, both cell lysates produced from E. coli carrying pRS21 (lane 2) or pCT8B (lane 3) showed identical immunoreactive protein bands with molecular weight of 68 kd, a sign that both are producing the same protein ( Figure 10) .
  • Plasmid pCTll.10 was constructed by cloning the 4.9 kb
  • the transcription direction of the collagenase gene is the same as the Lac promoter.
  • E. coli DH5 ⁇ carrying plasmid pCTll.10 was grown in 50 ml of TYE-Ap broth with or without ImM IPTG overnight at 32°C with shaking (130rpm) .
  • the cells and supernatant were separated by centrifugation.
  • the cells and supernatant were incubated with loading buffer at 95 ⁇ C before running on 7.5% SDS polyacrylamide gel.
  • Protein profiles of cell extracts are shown in lanes 1 (with IPTG) and 2 (without IPTG) of Figure 9. Protein profiles of culture media are shown in Lanes 3 (with IPTG) and 4 (without IPTG) of Figure 9.
  • the accumulation of collagenase in the culture media indicated that the cells excreted collagenase into the media from E. coli cells. These cells provide the advantage of a simple and cost effective method to produce and purify collagenase from culture media in which these host cells comprising the recombinant gene segment of the present invention coding for collagenase have grown.
  • E. coli strain DH5 ⁇ containing plasmid P70 was grown in TYE-Tc broth at 35°C for 5 hours with shaking. Cells were collected by centrifugation. Cell pellets were resuspended in 30 mM Tris-HCl, pH 8.0, 20% sucrose buffer. Lysozyme (70 micrograms/ml) and EDTA (2mM) were added and incubated at 4°C for 30 minutes to decompose the cell wall. The periplasmic fraction was separated from cytoplasmic and membrane fractions (CM fraction) by centrifugation.
  • CM fraction cytoplasmic and membrane fractions
  • periplasmic fraction and CM fraction were examined on SDS- PAGE and detected by both Coomossie blue staining and immunoblot methods. As shown in Figure 11, panel A, a distinct protein band with MW of 125kd was seen in the periplasmic fraction (lane 1) and barely seen in the cytoplasmic fraction (lane 2) on the Coomossie blue stainned gel.
  • the 68kd protein resides preferably in the cytoplasmic region, and not in the periplasmic region. This suggests that the 68 kd collagenase cannot be efficiently transported through the inner membrane. Although it was not clear that this 68 kd protein was identical to the 68 kd protein produced in pRS21, the 68 kd protein produced in pRS21 was not able to secrete into the periplasmic region. These data show that collagenase with MW of 125 kd produced in E. coli can utilize E. coli's secretion mechanism to transport through the inner membrane to accumulate in the periplasmic region.
  • EXAMPLE 10 Collagenase Production and Purification From E. coli cells and culture medium.
  • E. coli strain W3110 carrying plasmid P70 was grown in New Brunswick BioFlo III fermentor to produce collagenase.
  • the fermentation media (FM, grams per liter of media) consists of: KH2P04, 3.5; K2HP04, 5.0;
  • ZnCl 2 were prepared and autoclaved separately and 20 ul of each per liter were added.
  • E. coli strain carrying P70 was grown in TYE-Tc overnight at 30°C. The overnight culture was directly innoculated in the fermentor. After cell density reached OD600 equals to 16, the cells were harvested by centrifugation and separated from the supernatant.
  • the collagen digestion activity of the recombinant collagenase of the present invention either purified from cells or purified from culture medium was determined. The activity was measured as described below in Example 11. Table 1 below summarizes these activities.
  • Collagenase Produced by C. histolyticum A Size The identity of recombinant collagenase was compared to the native collagenase produced by C. histolyticum. Both unpurified and purified native forms of collagenase were purchased from Sigma, Boehringer Mannheim Products (Collagenase A, (CHSA) , Collagenase B (CHSB) , Collagenase D (CHSD) ) , Worthington, and CalbioChem. Different sources of collagenase were run on the SDS-PAGE to examine their molecular weight. A typical Coomossie blue stained gel and immunoblot is shown in Figure 6. Figure 6 shows comparisons between recombinant collagenase (RCL) and native collagenase; panel A is a Coomassie blue stained gel; panel B is a Western blot of SDS-PAGE.
  • the lane assigments for Figure 6 are as follows: Panel A: Lane 1, 0.5 ug of Lot 17 RCL; Lane 2, 2.5 ug of Lot 17 RCL; Lane 3, 2.5 ug of CHSD (BMB) ; Lane 4, 10 ug of CHSD; Lane 5, 2.5 ug of CHSB; Lane 6, 10 ug of CHSB; Lane7, 2.5 ug of CHSA; and Lane 8, 10 ug of CHSA.
  • Panel B Lane 1, 0.01 ug of Lot 17 RCL; Lane 2, 0.05 ug of Lot 17 RCL; Lane 3, 0.2 ug of CHSD; Lane 4, 0.2 ug of CHSB; and Lane 5, 0.5 ug of CHSA.
  • Purified recombinant collagenase of the present invention was compared with commercially available collagensaes obtained from Boehringer Mannheim Co. , catalog numbers CHSA, CHSB, and CHSD. Different amounts of proteins were run on the 7.5% SDS polyacrylamide gel. The collagenases on the Western blots were detected with rabbit anti-collagenase antibodies. Preparation of the anti ⁇ collagenase antibodies was described in U.S. Serial No. 07/498,919. Although a lot of proteins can be visualized by
  • the collagen digestion activities of commercially available purified native collagenase from different vendors were compared to the purified recombinant collagenase having molecular weight of 125 kd.
  • Collagen digestion activity was measured according to Mendl, I, et al, J.Clin. Invest. 32:1323 (1953) with modifications. The major modifications are 1) . to reduce the amount of enzyme used down to 0.2 or 0.4 ug and 2). to increase the substrate (Type 1 collagen) concentration to 10 mg/ml.
  • the collagenase activity is defined as the following: One unit of collagenase activity equals to one umole of L-leucine equivalents released from collagen after 5 hours of incubation at 37°C.
  • the enzyme activities of different purified collagenases are shown in Table 2 below.
  • the collagen digestion activity of the recombinant collagenase was about 25% to 100% higher than that of the native form of purified collagenases. The result clearly suggest that the recombinant collagenase of the present invention was superior to native collagenase.
  • recombinant collagenase of the present invention produced in E. coli was used to harvest endothelial cells from human saphenous veins.
  • a human saphenous vein was divided in half and perfused one half with 0.1% type II collagenase (Worthington), 0.5% BSA, and PBS/CMF and the other half with 0.048% recombinant collagenase in the same buffer with or without 0.01% trypsin.
  • Recombinant collagenase alone did not release endothelial cells from the vein.
  • RCL and trypsin the yield of endothelial cells doubled as compared to the crude collagenase.
  • Purified recombinant collagenase having a molecular weight of about 110,000 daltons (SEQUENCE ID NO. 2) and produced in E. coli was used to harvest rat islets from rat pancreas according to a method of the present invention.
  • Native class I and II collagenases and neutral protease were prepared according to G.H.J. Wolters, "An Analysis of the Role of Collagenase and Protease in the Enzymatic Dissociation of the Rat Pancreas for Islet Isolation," Diabetologia. 35:735-742 (1992).
  • Rat pancreas digestion was carried out as in Wolters (1992) .
  • a mixture comprising recombinant collagenase (SEQUENCE ID NO. 2) , native class II collagenase and neutral protease produced the same rat islet yield as compared to using the crude collagenase preparation or a combination of purified native collagenase and neutral protease.
  • This mixture comprised per 10 ml of KRH buffer (Wolters, 1992) 2 mg of class I recombinant collagenase (SEQUENCE ID NO. 2), 0.8 mg class II native collagenase, and 100 units of neutral protease. Similar digestion time required to obtain the same islet yields were also observed.
  • compositions comprising the highly purified recombinant collagenase (SEQUENCE ID NO. 2) can be used in the method of the present invention for islet isolation from humans.
  • MOLECULE TYPE DNA (genomic)
  • GGT GTA AGT ACT GAT AAT GGT GGT CTA TAT ATA GAA CCA AGA GGG ACT 96 Gl ⁇ OVal Ser Thr Asp Asn Gly Gly Leu Tyr He Glu Pro Arg Gly Thr 305 310 315 320
  • AA_A5GAG GGA GAC GAT CAA AAT CAT ATT GCA AGT GGT ATA GAT AAG AAT 235 Lys Glu Gly Asp Asp Gin Asn His He Ala Ser Gly He Asp Lys Asn 770 775 780 AAC TCA AAA GTT GGA ACA TTT AAA GCT ACA AAA GGA AGA CAT TAT GTG 2400 Asn Ser Lys Val Gly Thr Phe Lys Ala Thr Lys Gly Arg His Tyr Val 785 790 795 800
  • TCTOTCT GAT AAA GCT ACA GTT ATA CCA AAT TTC AAT ACC ACT ATG CAA 2544 Ser Asp Lys Ala Thr Val He Pro Asn Phe Asn Thr Thr Met Gin 835 840 845
  • MOLECULE TYPE DNA (genomic)

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Abstract

E. coli manipulé génétiquement portant des vecteurs qui contiennent un segment d'insertion codant pour des formes natives et non natives de la collagénase de Clostridium histolyticum. Les segments d'ADN recombiné codant pour la collagénase sont exprimés de manière efficace dans les cellules transformées, de manière à obtenir une collagénase à activité enzymatique et à réactivité croisée immunologique. La collagénase manipulée génétiquement sert à isoler les îlots pancréatiques, à isoler les cellules endothéliales, à isoler les cellules tumorales dispersées, et à traiter la hernie discale.
PCT/US1993/005944 1992-06-22 1993-06-22 CLONAGE MOLECULAIRE DES GENES RESPONSABLES DE LA PRODUCTION DE COLLAGENASE A PARTIR DE $i(CLOSTRIDIUM HISTOLYTICUM) WO1994000580A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP93916670A EP0654084A4 (fr) 1992-06-22 1993-06-22 CLONAGE MOLECULAIRE DES GENES RESPONSABLES DE LA PRODUCTION DE COLLAGENASE A PARTIR DE -i(CLOSTRIDIUM HISTOLYTICUM).
JP6502521A JPH08500970A (ja) 1992-06-22 1993-06-22 ヒストリチクス菌からのコラゲナーゼ産生に応答性の遺伝子の分子クローニング
AU46447/93A AU4644793A (en) 1992-06-22 1993-06-22 Molecular cloning of the genes reponsible for collagenase production from clostridium histolyticum

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US90212992A 1992-06-22 1992-06-22
US07/902,129 1992-06-22

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WO1994000580A1 true WO1994000580A1 (fr) 1994-01-06

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EP (1) EP0654084A4 (fr)
JP (1) JPH08500970A (fr)
AU (1) AU4644793A (fr)
CA (1) CA2138948A1 (fr)
WO (1) WO1994000580A1 (fr)

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WO1995005477A1 (fr) * 1993-08-12 1995-02-23 University Of Maryland Metalloprotease alcaline thermostable secretee par un hyphomonas et sa preparation
EP0677586A1 (fr) * 1994-03-16 1995-10-18 Boehringer Mannheim Gmbh Collagenase type II de clostridium histolyticum et son utilisation pour l'isolation de cellules et d'agglomérations cellulaires
WO1996028543A1 (fr) * 1995-03-16 1996-09-19 Knoll Aktiengesellschaft Nouveaux melanges enzymatiques definis pour l'obtention de cellules et pour le traitement de blessures
WO1996034093A1 (fr) * 1995-04-25 1996-10-31 Baxter International Inc. Composition contenant de la collagenase et la chymopapaine pour isoler des hepatocytes et des cellules d'ilots pancreatiques
US5670358A (en) * 1995-10-19 1997-09-23 Baxter International Inc. Method for inhibiting chymopapain and papain enzyme activity with polysaccharides of animal origin
WO1998022574A2 (fr) * 1996-11-19 1998-05-28 Roche Diagnostics Gmbh Collagenase recombinee type i provenant du clostridium histolyticum, et son utilisation pour l'isolation des cellules et des agglomerations cellulaires
US6280993B1 (en) * 1999-08-24 2001-08-28 Ichiro Yamato Gene encoding class I collagenase
ITRM20090661A1 (it) * 2009-12-15 2011-06-16 Federico Bertuzzi Collagenasi ricombinanti di c. histolyticum e metodo per la loro produzione.
CN102174548A (zh) * 2011-03-01 2011-09-07 山东大学 一种深海适冷耐盐胶原蛋白酶及其编码基因myr02与应用
US9757435B2 (en) 2012-01-12 2017-09-12 Endo Global Ventures Clostridium histolyticum enzymes and methods for the use thereof
US11123280B2 (en) 2017-03-01 2021-09-21 Endo Ventures Limited Method of assessing and treating cellulite
US11473074B2 (en) 2017-03-28 2022-10-18 Endo Global Aesthetics Limited Method of producing collagenase

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US7811560B2 (en) * 2006-01-30 2010-10-12 Auxilium Us Holdings, Llc Compositions and methods for treating collagen-mediated diseases
US20090191608A1 (en) * 2008-01-22 2009-07-30 Baylor Research Institute Pancreatic Islet Cell Preparation and Transplantation
CN115058443B (zh) * 2022-06-22 2023-03-24 广州市乾相生物科技有限公司 转录抑制质粒pCRISPR-nadG及其在提高产溶剂梭菌胞内还原力中的应用

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Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995005477A1 (fr) * 1993-08-12 1995-02-23 University Of Maryland Metalloprotease alcaline thermostable secretee par un hyphomonas et sa preparation
EP0677586A1 (fr) * 1994-03-16 1995-10-18 Boehringer Mannheim Gmbh Collagenase type II de clostridium histolyticum et son utilisation pour l'isolation de cellules et d'agglomérations cellulaires
EA000583B1 (ru) * 1995-03-16 1999-12-29 Кноль Аг СМЕСЬ ФЕРМЕНТОВ, ВЫДЕЛЕННЫХ ИЗ БАКТЕРИЙ Clostridium histolyticum
WO1996028543A1 (fr) * 1995-03-16 1996-09-19 Knoll Aktiengesellschaft Nouveaux melanges enzymatiques definis pour l'obtention de cellules et pour le traitement de blessures
US6146626A (en) * 1995-03-16 2000-11-14 Knoll Aktiengesellschaft Defined enzyme mixtures for obtaining cells and treating wounds
WO1996034093A1 (fr) * 1995-04-25 1996-10-31 Baxter International Inc. Composition contenant de la collagenase et la chymopapaine pour isoler des hepatocytes et des cellules d'ilots pancreatiques
US5670358A (en) * 1995-10-19 1997-09-23 Baxter International Inc. Method for inhibiting chymopapain and papain enzyme activity with polysaccharides of animal origin
US6475764B1 (en) 1996-11-19 2002-11-05 Roche Diagnostics Gmbh Recombinant collagenase type I from clostridium histolyticum and its use for isolating cells and groups of cells
WO1998022574A2 (fr) * 1996-11-19 1998-05-28 Roche Diagnostics Gmbh Collagenase recombinee type i provenant du clostridium histolyticum, et son utilisation pour l'isolation des cellules et des agglomerations cellulaires
WO1998022574A3 (fr) * 1996-11-19 1998-07-09 Boehringer Mannheim Gmbh Collagenase recombinee type i provenant du clostridium histolyticum, et son utilisation pour l'isolation des cellules et des agglomerations cellulaires
US6280993B1 (en) * 1999-08-24 2001-08-28 Ichiro Yamato Gene encoding class I collagenase
US8715985B2 (en) 2009-12-15 2014-05-06 Abiel S.R.L. Clostridium histolyticum recombinant collagenases and method for the manufacture thereof
ITRM20090661A1 (it) * 2009-12-15 2011-06-16 Federico Bertuzzi Collagenasi ricombinanti di c. histolyticum e metodo per la loro produzione.
WO2011073925A2 (fr) 2009-12-15 2011-06-23 Abiel S.R.L. Collagénases de recombinaison de c. histolyticum et procédé de production de ces dernières
WO2011073925A3 (fr) * 2009-12-15 2011-08-11 Abiel S.R.L. Collagénases de recombinaison de c. histolyticum et procédé de production de ces dernières
CN102174548A (zh) * 2011-03-01 2011-09-07 山东大学 一种深海适冷耐盐胶原蛋白酶及其编码基因myr02与应用
CN102174548B (zh) * 2011-03-01 2012-07-11 山东大学 一种深海适冷耐盐胶原蛋白酶及其编码基因myr02与应用
US9757435B2 (en) 2012-01-12 2017-09-12 Endo Global Ventures Clostridium histolyticum enzymes and methods for the use thereof
US11879141B2 (en) 2012-01-12 2024-01-23 Endo Global Ventures Nucleic acid molecules encoding clostridium histolyticum collagenase II and methods of producing the same
US11975054B2 (en) 2012-01-12 2024-05-07 Endo Global Ventures Nucleic acid molecules encoding clostridium histolyticum collagenase I and methods of producing the same
US11123280B2 (en) 2017-03-01 2021-09-21 Endo Ventures Limited Method of assessing and treating cellulite
US11813347B2 (en) 2017-03-01 2023-11-14 Endo Ventures Limited Method of assessing and treating cellulite
US11473074B2 (en) 2017-03-28 2022-10-18 Endo Global Aesthetics Limited Method of producing collagenase

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EP0654084A1 (fr) 1995-05-24
EP0654084A4 (fr) 1996-07-03
CA2138948A1 (fr) 1994-01-06
JPH08500970A (ja) 1996-02-06
AU4644793A (en) 1994-01-24

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