AU4644793A - Molecular cloning of the genes reponsible for collagenase production from clostridium histolyticum - Google Patents

Description

MOLECULAR CLONING OF THE GENES

RESPONSIBLE FOR COLLAGENASE PRODUCTION

FROM CLOSTRIDIUM HISTOLYTICUM

BACKGROUND 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. In particular, 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.

SUMMARY

In accordance with the present invention, a recombinant DNA segment is provided which codes for a polypeptide having the enzymatic activity and antigenicity of Clostridium histolvticiu collagenase. This polypeptide, however, 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 inventors found that the expression of the claimed recombinant DNA segment in transformed host cells is determined by the strain of the host cell. Accordingly, different E. coli host cells transformed with the vector of the invention are provided. One strain of host cells produces the non-native polypeptide of the invention having collagenase activity and antigenicity. Greater than 50% by weight of the total polypeptides produced by the host cells that have collagenase activity comprise the non-native high molecular weight 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:

(1) incubating the tissue to be dispersed in a buffered solution containing the substantially purified genetically engineered collagenases of the present invention with shaking at about 25-39° C to release and disperse the embedded cells; and

(2) separating the dispersed cells from tissue debris. 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:

(1) preparing a sterile buffered solution containing the claimed substantially purified genetically engineered collagenase; and

(2) injecting the sterile buffered solution containing the substantially purified claimed collagenase into the nucleus pulposus.

These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description, appended claims, and the accompanying figures. FIGURES

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 DH5α.

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 DH5α 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 DH5α 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. DETAILED DESCRIPTION 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. Depending on the E. coli host into which it was inserted, it was found that the same recombinant DNA segment, having 4.9 kilobases, could be translated to yield several polypeptides having collagenase activity, as described below in Examples 3 and 11. E. coli strain DH5α, 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. C. 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. As described in the Examples below, it was found that 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. It was found, as detailed in Example 8, that the claimed recombinant DNA segment comprised a promoter derived from C. histolyticum. As described below, 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. As described in the Examples, 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. After DNA was isolated from C. histolyticum. 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) . 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.

The expression of 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. Using standard techniques to achieve transformation and collagenase detection as described in U.S.Application Serial No. 07/498,919, 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.

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"). As presented in Example 12, the purified 110 kd recombinant collagenase of the invention, in combination with trypsin, was effective for isolating endothelial cells from human saphenous veins.

Depending on the targeted tissue and animal, 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. (G.H.J. Wolters, "An Analysis of the Role of Collagenase and Protease in the Enzymatic Dissociation of the Rat Pancreas for Islet Isolation." Diabetoioσia. 35:735-742 (1992)).

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.

EXAMPLES The following examples are for illustrative purposes only and are not to be construed as limiting the invention.

Example 1 Cloning and Screening of the Gene Encoding

For the Intact 125kd Collagenase

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. An attempt was made to clone the Bglll fragment directly from genomic DNA to small plasmids like pBluescript. The inventors identified a collagenase band at MW of 125 kd, but upon a second screening, involving two independent experiments, the molecular weight appeared to be 68 kd.

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^βC 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 μg/mL, respectively. 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.

B. Construction of a pRK290 library containing clostridium DNA Purified C. histolyticum chromosomal DNA was digested with restriction enzyme Bglll. After ethanol precipitation, the digested DNA was resuspended in TE buffer (10 mM Tris, pH 7.4, 0.1 mM EDTA). Appropriate amounts of digested DNA were ligated with pRK290 that had been previously cleaved with Bglll and treated with alkaline phosphatase. The ligation was performed at 4°C for 16 hours. The ligated DNA was transformed into E. coli strain DH5α as described. Transformed cells were plated on TYE-Tc (15 g tryptone, 10 g yeast extract, 5 g NaCl, and 15 mg of tetracycline per liter) plate and incubated at 35°C overnight.

C. Screening for Collagenase Gene

Approximately 1,000 colonies were obtained per plate. About 10 plates were screened for intact collagenase gene. Briefly, after 16 hours of incubation at 35°C, a dried nitrocellulose paper (S&S) was overlayed on the petri dish to absorb the colonies. The nitrocellulose paper was carefully separated from the plates, transferred to a freshly prepared TYE-Tc agar plate, and incubated for another 4 hours at 35°C. The original plates were stored as master plates at 4"C. The nitrocellulose paper was carefully lifted from the plate, soaked in the 0.1M Tris, pH 8.0, 0.2 M NaCl, and 5% skim milk, and shaken gently to remove the bacteria cells from the 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, plϊ 8.0, containing 0.2 M NaCl and 1% Triton™ 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 H₂0₂ 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).

The area of agarose on the plate corresponding to the location of a positive signal on the nitrocellulose was removed and resuspended in TYE-Tc broth. Serially diluted cell suspension to low cell densities (30-100 cells per plate) were replated on the TYE-Tc plates and a second screening was performed to identify single colony. One colony designated P70 showed a strong signal and was collected and grown in TYE-Tc broth. This colony was further characterized as below in Examples 2 and 3.

EXAMPLE 2

Restriction Enzyme Analysis of Inserted DNA

To characterize the organization of the Clostridium

DNA in plasmid P70 more precisely, 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. A

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.

Both P70 and pBBl releaesd an identical 0.8 kb EcoRI-EcoRI fragment. These restriction enzyme analysis suggested that all three plasmids share common DNA fragment and P70 carries an extra piece of 2.4 kb Bglll-EcoRI fragment.

EXAMPLE 3 Characterization of Collagenase Produced in E. coli Strain DHSct To examine the size of immunoreactive protein, cells containing P70 were collected by centrifugation, resuspended in gel loading buffer, heated, and run on a 7.5% SDS-Polyacrylamide gel as described in Laemmli, U.K., "Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4", Nature 227:680-685 (1970). The proteins were electroblotted to nitrocellulose paper and detected with the rabbit anticollagenase antibodies of U.S. Application Serial No. 07/498,919 (W.H. Burnette, "Western Blotting: Electrophoretic Transfer of Proteins from SDS- polyacrylamide Gels to Unmodified Nitrocellulose and Radiographic Detection with Antibody and Radioiodinated Protein A" Anal. Biochem. 112. 195-203 (1981Ϊ . The molecular weight of the collagenase from the P70 clone was determined by comparison with prestained protein standards. Immunoreactive protein bands with molecular weight of approximately 125 kd and downward were expressed from the P70 clone. Several bands with molecular weights between 68 and 100 kd are similar to what we described in U.S. Application Serial No. 07/498,919.

E. coli strain DH5α 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.

The expression of the recombinant DNA segment of the present invention is shown in Figure 2. Panel A is Coomassie blue-stainned SDS-PAGE. Lane 1, E. coli strain DH5α carrying plasmid pUC8 and Lane 2, E. coli strain DH5α 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.

In the cell extracts prepared from cells carrying plasmid P70 but not from cells carrying pUC8, a very strong Coomassie blue stained protein band was visualized between molecular weights 200 and 97 kilodaltons. Plasmid P70 transformed E. coli strain DH5α 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.

EXAMPLE 4 Characterization of the Collagenase Gene

A. DNA sequencing strategy.

A non-random DNA sequencing strategy published by Henikoff, S. (Methods in Enzymologγ. Unidirectional Digestion With Exonuclease III in DNA Sequence Anallysis, 155:156 (1987)) with minor modification was used to sequence the 2.5 kb EcoRI-Bglll DNA fragment encoded by pBBl. Plasmids pCT6 and pCT7 were constructed by inserting the 2.5 kb BamHI/Bglll fragment into the BamHI site of plasmid pBluescript SK(-) in the opposite orientation. 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 3¹- 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 DH5α. After transformation, the cells were plated on TYE-Ap plates, and grown overnight at 35°C. To examine the deletion and prepare DNA for sequencing, 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. After RNase digestion, the DNA was precipitated with 10 PEG and 1.25M NaCl. DNA pellets were washed with 100% ethanol and resuspended in TE buffer. DNA prepared by this method was adequate for DNA sequencing using Sanger¹s dideoxy chain termination method (Sanger, F. and A.R. Coulson, J. Molec. Biol. 94:441 (1975)). Clones used for DNA sequencing and the sequence information obtained are summarized in Figure 3. In some occasions, plasmid pCT12.8 was systematically deleted and subclones were used to determine part of the sequences. Plasmid pCT12.8 was constructed by inserting the 4.9 kb Bglll fragment of P70 into pBluescript (SK-) pretreated with BamHI.

B. DNA sequence and analysis

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. editors, page 123, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1983)) was identified 11 base pairs downstream from the translational termination signal (TAA) as shown in Figure 5. This signal consists of a stem-loop structure followed by an A-T rich sequence. This data suggested that the collagenase gene ends in the A-T rich area downstream of the stem-loop structure.

EXAMPLE 5

DNA Sequence Comparison of Previous Clones Although it is clear that all the previous clones produced identical immunoreactive protein bands as judged by their mobilities on the immunoblots, it did not rule out the possibility of microheterogeneity. If microheterogeneity existed among the clones obtained previously, it may have supported the notion proposed by Van Wart et al [REF] that gene duplication and then mutation produced different collagenases. To confirm their identities, two DNA sequence projects were carried out: 1) . Comparison of sequences between clones with different inserts such as P6, P9, P41, P42, and P51 and 2) Comparison of sequences among different original isolates obtained previously, all apparently having the same insert size. DNA sequences flanked by multiple cloning sites were sequenced using the flanking universal primers. An average of 150 to 200 base pairs of Clostridium DNA sequence were obtained from both ends. After comparing the sequences, no single base pair differences was identified. This result did not support gene duplication as the means responsible for obtaining differences in the same class of collagenase. EXAMPLE 6 Comparison of the DNA Sequence of Clones Derived From P70 to Sequences from Cloness Obtained Previously To examine the identities of P70 and other clones obtained previously, many clones were partially sequenced and compared. The 0.7 kb EcoRI and 1.8 kb EcoRI-Bglll fragments from clones P70 were subcloned into pUC13 or pBluescript. 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.

EXAMPLE 7

Comparison of Collagenase Produced from Subclone of P70

To the Collagenase Produced from pRS21

To further characterize the identity between P70 and the previously described clones, the collagenase products produced from subclones apparently having the same inserts were compared. p70 was digested with Bglll and EcoRI and ligated with pUClδ predigested with BamHI and EcoRI. Ligated DNA was transformed to E. coli strain DH5α, plated on TYE-Ap plate, and incubated at 35° C overnight. A clone containing the 1.7kb EcoRI-Bglll insert were and having the same insert orientation as pRS21 was identified and designated as pCT8B. E. coli strain DH5α 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 pUClδ 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) .

EXAMPLE 8 Control of Collagenase Expression in E. Coli By Independent Promoter Derived from Clostridium Histolyticum

Plasmid pCTll.10 was constructed by cloning the 4.9 kb

Bglll fragment from P70 into plasmid pUC13 that was predigested with BamHI and is shown in Figure 8. The transcription direction of the collagenase gene is the same as the Lac promoter.

E. coli DH5α 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^βC before running on 7.5% SDS polyacrylamide gel. Twenty ul and 80 ul equivalent to the original volume of supernatant and cells, respectively, were loaded per lane and shown in Figure 9. After Coomassie blue stainning, 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.

With or without IPTG, both cell extracts and culture media did not show any expression differences of recombinant collagenase. IPTG, a potent lac promoter inducer, did not increase the expression of collagenase, which further confirmed that the 4.9 kb DNA fragment contained a Clostridium promoter that could function independently in E. coli. Unexpectedly, a large amount of collagenase was discovered in the supernatant as shown in Lanes 3 and 4 of Figure 9. Since the equivalent volume of supernatant loaded (20 ul) was one fourth of the equivalent volume of cells loaded (80 ul) , it was estimated that 80% of collagenase resided in the supernatant, as judged from the stained gel. 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.

EXAMPLE 9 Intracellular Localization of Collagenases Expressed in

E. coli

The intracellular localization of collagenase in different E. coli compartment was examined. E. coli strain DH5α 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. The 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.

Many immunoreactive bands were seen by the immunoblot as shown in Panel B of Figure 11. Most of the bands existed in the periplasmic region (lane 1 of Panel B) , which suggested that the protein represented by these bands can secrete through the inner membrane and accumulate in the periplasmic space. Based on the amount loaded on the gel (the periplasmic fraction was loaded at half the equivalent original volume as compared to the CM fraction) , it is estimated that 80% of the 125 kd collagenase was located in the periplasmic region. Although the ratio of immunoreactive bands residing in the periplasmic and cytoplasmic regions (lane 2) are different among the immunoreactive bands, most of these bands apparently are preferably located in the perplasmic region except for a protein band with MW of 68kd. 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.

A. Production of Collagenase from E. coli

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;

(NH4)2HP04, 3.5; MgS04.7H20, 3.5; Yeast Extract, and 5;

Tryptone, 5. 50% of glucose was autoclaved separately and

10 ml per liter was added. 1M stock solutions of CaCl₂ and

ZnCl₂ 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.

B. Purification from Cells

Cells were collected by centrifugation and resuspended in lOmM Bis-Tris buffer, pH 6.5, with ImM PMSF. The cells were disrupted by sonication and debri were removed by centrifugation. Proteins were fractionated by sequential ammonium sulfate precipitation. Fractions containing collagenase were resuspended in lOmM Bis-Tris buffer (Buffer A) and dialyzed against the same buffer overnight at 4°C. Dialyzed samples were first fractionated by DE52 column chromatography. Fractions containing collagenase were pooled and dialyzed against buffer A. Dialyzed samples were fractionated by Q Sepharose column chromatography. To obtain greater than 98% of purity, gel filtration may be required. Many immunoreactive collagenase bands co-existed in the starting material. Different molecular weight collagenase could be separated with anion exchange column chromatography to a certain extent. The experiment described below in Example li used the largest form of collagenase. C. Purification from Media Unexpectedly, a large amount of collagenase was detected in the fermentation broth. To evaluate the possibility of collecting and purifying collagenase from culture media, a purification strategy was developed. Briefly, after the cells were removed by centrifugation, the supernatant was concentrated and diafiltrated using hollow fiber with molecular weight cut-off of 30,000 (Amicon) . Concentrated supernatant was fractionated by ammonium sulfate differential precipitation. Fractions containing collagenase were collected, resuspended in Bis- Tris Buffer, pH6.5, and dialyzed against the same buffer. Dialyzed sample was loaded onto the DE52 column (Whatman) and eluted with NaCl. Fractions containing the collagenase were collected and further purified on the Q Sepharose column (Pharmacia). Figure 7, lane 1 shows a Coomassie blue stained gel of culture media from E. coli strain W3110 carrying plasmid P70 (Lane 1) and collagenase purified from this culture media (Lane 2) . Collagen digestion activities of collagenase prepared from both cells and culture medium were compared. Collagenase prepared from medium had about two times the activity as that prepared from cells.

D. Collagen Digestion Activity of Collagenase Purified from Cells and Medium.

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.

Table 1. Collagenase Activity or Recombinant Collagenase Purified From Cells or Purified from Medium

The results of this table show that the collagenase activity purified from the medium is about equivalent to the activity of the recombinant collagenase purified from cells.

22

SUBSTITUTE SHEET EXAMPLE 11 Comparisons of Recombinant Collagenase and Native

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

Coomossie blue staining, more than 50% of the stainable bands can not be detected by collagenase specific antibodies. The largest form of recombinant collagenase clearly shows a larger size than any of the native forms of collagenase obtained from Boehringer Mannheim. The identical result was obtained in comparing the recombinant collagenase of the present invention to the native collagenases obtained from Calbiochem, Worthington, and Sigma.

A form of recombinant collagenase similar to the largest collagenase (llOkd) of native products could be produced quantitatively and consistently in a special E. coli host (Strain JM105) . B. Activity

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.

Table 2. Comparisons of Collagen Digestion Activities among Different Purified Collagenases

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.

EXAMPLE 12 Endothelial Cell Isolation With Recombinant Collagenase

Plus Trypsin Purified 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. By combining RCL and trypsin, the yield of endothelial cells doubled as compared to the crude collagenase. These results demonstrated that more endothelial cells can be isolated by combining recombinant collagenase of the present invention with a constant concentration of trypsin. These results further suggested that it was possible to replace native collagenase with recombinant collagenase of the present invention for cell isolation from different tissues.

25

SUBSTITUTE SHEET EXAMPLE 13 Rat Islet Isolation Using Enzyme Mixture Comprising Recombinant Collagenase A composition comprising recombinant collagenase (SEQUENCE ID NO. 2) , native class II collagenase, and neutral proteases was developed to disperse pancreatic tissue to release islets.

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.

These results demonstrated that a composition which comprised the substantially purified recombinant collagenase (SEQUENCE ID NO. 2) dispersed pancreatic tissue to release islets from other pancreatic components. Furthermore, it was observed that fewer islets each having a larger islet mass per islet resulted when pancreases were digested using a composition comprising recombinant collagenase (SEQUENCE ID NO. 2) and other proteases as compared to other combinations described in the Wolters, 1992 reference. It appeared that digesting pancreases using the method of the present invention resulted in less damage to the integrity of islets as compared to using enzyme compositions which did not comprise the recombinant collagenase (SEQUENCE ID NO. 2) of the present invention.

It is known that larger animals require different lots of crude collagenase preparations to obtain satisfactory islets from pancreas digestion. Accordingly, the amounts of recombinant collagenase (SEQUENCE ID NO. 2) , or native class II collagenase, or neutral proteases can be varied to obtain pancreatic islet preparation from pancreas digestions of other animals. Protocols for determining the concentrations of proteolytic enzymes useful for tissue digestions are exemplified in Wolters, 1992. 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.

Although the present invention has been described in considerable detail with regard to certain preferred versions thereof, other versions are possible. Therefore, the spirit and scope of the amended claims should not be limited to the descriptions of the preferred versions herein.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Lin et al., Hun-Chi

(ii) TITLE OF INVENTION: Molecular cloning of the genes 5 responsible for collagenase product

(iii) NUMBER OF SEQUENCES: 3

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Harris Brotman

10 (B) STREET: 401 B. St Ste 1700

(C) CITY: San Diego

(D) STATE: CA

(E) COUNTRY: USA

(F) ZIP: 92101-4297

15 (V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

20 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: 25 (A) NAME: Brotman, Harris F.

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (619) 699-3630

(B) TELEFAX: (619) 236-1048 ( 2 ) INFORMATION FOR SEQ ID N0 : 1 :

( i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 2817 base pairs

(B) TYPE : nucleic acid

5 (C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS 10 (B) LOCATION: 1..2808

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:

ATG AAG GGT ATA GAA ACT TTC ACT GAG GTT TTA AGA GCT GGT TTT TAT 4 Met Lys Gly lie Glu Thr Phe Thr Glu Val Leu Arg Ala Gly Phe Tyr 1 5 10 15

TTA5GGG TAC TAT AAT GAT GGT TTA TCT TAT TTA AAT GAT AGA AAC TTC 9 Leu Gly Tyr Tyr Asn Asp Gly Leu Ser Tyr Leu Asn Asp Arg Asn Phe 20 25 30

CAA GAT AAA TGT ATA CCT GCA ATG ATT GCA ATT CAA AAA AAT CCT AAC 14 Gin Asp Lys Cys lie Pro Ala Met lie Ala lie Gin Lys Asn Pro Asn 20 35 40 45

TTT AAG CTA GGA ACT GCA GTT CAA GAT GAA GTT ATA ACT TCT TTA GGA 19 Phe Lys Leu Gly Thr Ala Val Gin Asp Glu Val lie Thr Ser Leu Gly 50 55 60

AAA CTA ATA GGA AAT GCT TCT GCT AAT GCT GAA GTA GTT AAT AAT TGT 24 Lys Leu lie Gly Asn Ala Ser Ala Asn Ala Glu Val Val Asn Asn Cys 65 70 75 80

GTA CCA GTT CTA AAA CAA TTT AGA GAA AAC TTA AAT CAA TAT GCT CCT 28 ValδPro Val Leu Lys Gin Phe Arg Glu Asn Leu Asn Gin Tyr Ala Pro 85 90 95

GAT TAC GTT AAA GGA ACA GCT GTA AAT GAA TTA ATT AAA GGT ATT GAA 33 Asp Tyr Val Lys Gly Thr Ala Val Asn Glu Leu lie Lys Gly lie Glu 100 105 110

TTEOGAT TTT TCT GGT GCT GCA TAT GAA AAA GAT GTT AAG ACA ATG CCT 38 Phe Asp Phe Ser Gly Ala Ala Tyr Glu Lys Asp Val Lys Thr Met Pro 115 120 125

TGG TAT GGA AAA ATT GAT CCA TTT ATA AAT GAA CTT AAG GCC TTA GGT 43 Trp Tyr Gly Lys lie Asp Pro Phe lie Asn Glu Leu Lys Ala Leu Gly 15130 135 140

CTA TAT GGA AAT ATA ACA AGT GCA ACT GAG TGG GCA TCT GAT GTT GGA 48 Leu Tyr Gly Asn lie Thr Ser Ala Thr Glu Trp Ala Ser Asp Val Gly 145 150 155 160

ATA TAC TAT TTA AGT AAA TTC GGG CTT TAC TCA ACT AAC CGA AAT GAC 52 IiaO yr Tyr Leu Ser Lys Phe Gly Leu Tyr Ser Thr Asn Arg Asn Asp 165 170 175

ATA GTA CAG TCA CTT GAA AAG GCT GTA GAT ATG TAT AAG TAT GGT AAA 57 lie Val Gin Ser Leu Glu Lys Ala Val Asp Met Tyr Lys Tyr Gly Lys 180 185 190

AT£5GCC TTT GTA GCA ATG GAG AGA ATA ACT TGG GAT TAT GAT GGG ATT 62 lie Ala Phe Val Ala Met Glu Arg lie Thr Trp Asp Tyr Asp Gly lie 195 200 205 GGT TCT AAT GGT AAA AAG GTG GAT CAC GAT AAG TTC TTA GAT GAT GCT 672 Gly Ser Asn Gly Lys Lys Val Asp His Asp Lys Phe Leu Asp Asp Ala 210 215 220

GAA AAA CAT TAT CTG CCA AAG ACA TAT ACT TTT GAT AAT GGA ACC TTT 720 GluSLys His Tyr Leu Pro Lys Thr Tyr Thr Phe Asp Asn Gly Thr Phe 225 230 235 240

ATT ATA AGA GCA GGG GAT AAG GTA TCC GAA GAA AAA ATA AAA AGG CTA 768 lie lie Arg Ala Gly Asp Lys Val Ser Glu Glu Lys He Lys Arg Leu 245 250 255

TATO GG GCA TCA AGA GAA GTG AAG TCT CAA TTC CAT AGA GTA GTT GGC 81 Tyr Trp Ala Ser Arg Glu Val Lys Ser Gin Phe His Arg Val Val Gly 260 265 270

AAT GAT AAA GCT TTA GAG GTG GGA AAT GCC GAT GAT GTT TTA ACT ATG 864 Asn Asp Lys Ala Leu Glu Val Gly Asn Ala Asp Asp Val Leu Thr Met 15 275 280 285

AAA ATA TTT AAT AGC CCA GAA GAA TAT AAA TTT AAT ACC AAT ATA AAT 91 Lys He Phe Asn Ser Pro Glu Glu Tyr Lys Phe Asn Thr Asn He Asn 290 295 300

GGT GTA AGT ACT GAT AAT GGT GGT CTA TAT ATA GAA CCA AGA GGG ACT 96 GlξOVal Ser Thr Asp Asn Gly Gly Leu Tyr He Glu Pro Arg Gly Thr 305 310 315 320

TTC TAC ACT TAT GAG AGA ACA CCT CAA CAA AGT ATA TTT AGT CTT GAA 100 Phe Tyr Thr Tyr Glu Arg Thr Pro Gin Gin Ser He Phe Ser Leu Glu 325 330 335

GAΛ5TTG TTT AGA CAT GAA TAT ACT CAC TAT TTA CAA GCG AGA TAT CTT 105 Glu Leu Phe Arg His Glu Tyr Thr His Tyr Leu Gin Ala Arg Tyr Leu 340 345 350 GTA GAT GGT TTA TGG GGG CAA GGT CCA TTT TAT GAA AAA AAT AGA TTA 11 Val Asp Gly Leu Trp Gly Gin Gly Pro Phe Tyr Glu Lys Asn Arg Leu 355 360 365

ACT TGG TTT GAT GAA GGT ACA GCT GAA TTC TTT GCA GGA TCT ACC CGT 11 Thr5Trp Phe Asp Glu Gly Thr Ala Glu Phe Phe Ala Gly Ser Thr Arg 370 375 380

ACA TCT GGT GTT TTA CCA AGA AAA TCA ATA TTA GGA TAT TTG GCT AAG 12 Thr Ser Gly Val Leu Pro Arg Lys Ser He Leu Gly Tyr Leu Ala Lys 385 390 395 400

GATOAAA GTA GAT CAT AGA TAC TCA TTA AAG AAG ACT CTT AAT TCA GGG 12 Asp Lys Val Asp His Arg Tyr Ser Leu Lys Lys Thr Leu Asn Ser Gly 405 410 415

TAT GAT GAC AGT GAT TGG ATG TTC TAT AAT TAT GGA TTT GCA GTT GCA 12 Tyr Asp Asp Ser Asp Trp Met Phe Tyr Asn Tyr Gly Phe Ala Val Ala 15 420 425 430

CAT TAC CTA TAT GAA AAA GAT ATG CCT ACA TTT ATT AAG ATG AAT AAA 13 His Tyr Leu Tyr Glu Lys Asp Met Pro Thr Phe He Lys Met Asn Lys 435 440 445

GCT ATA TTG AAT ACA GAT GTG AAA TCT TAT GAT GAA ATA ATA AAA AAA 13 A12_0Ile Leu Asn Thr Asp Val Lys Ser Tyr Asp Glu He He Lys Lys 450 455 460

TTA AGT GAT GAT GCA AAT AAA AAT ACA GAA TAT CAA AAC CAT ATT CAA 14 Leu Ser Asp Asp Ala Asn Lys Asn Thr Glu Tyr Gin Asn His He Gin 465 470 475 480

GAE5TTA GTA GAT AAA TAT CAA GGA GCT GGA CTA CCT CTA GTA TCA GAT 14 Glu Leu Val Asp Lys Tyr Gin Gly Ala Gly Leu Pro Leu Val Ser Asp 485 490 495 GAT TAC TTA AAA GAT CAT GGA TAT AAG AAA GCA TCT GAA GTA TAT TCT 153 Asp Tyr Leu Lys Asp His Gly Tyr Lys Lys Ala Ser Glu Val Tyr Ser 500 505 510

GAA ATT TCA AAA GCT GCT TCT CTT ACA AAC ACT AGT GTA ACA GCA GAA 158 Gluδlle Ser Lys Ala Ala Ser Leu Thr Asn Thr Ser Val Thr Ala Glu 515 520 525

AAA TCT CAA TAC TTT AAC ACA TTC ACT TTA AGA GGA ACT TAT ACA GGT 163 Lys Ser Gin Tyr Phe Asn Thr Phe Thr Leu Arg Gly Thr Tyr Thr Gly 530 535 540

GAAOACT TCT AAA GGT GAA TTT AAA GAT TGG GAT GAA ATG AGT AAA AAA 168 Glu Thr Ser Lys Gly Glu Phe Lys Asp Trp Asp Glu Met Ser Lys Lys 545 550 555 560

TTA GAT GGA ACT TTG GAG TCC CTT GCT AAA AAT TCT TGG AGT GGA TAC 172 Leu Asp Gly Thr Leu Glu Ser Leu Ala Lys Asn Ser Trp Ser Gly Tyr 15 565 570 575

AAA ACC TTA ACA GCA TAC TTT ACG AAT TAT AGA GTT ACA AGC GAT AAT 177 Lys Thr Leu Thr Ala Tyr Phe Thr Asn Tyr Arg Val Thr Ser Asp Asn 580 585 590

AAA GTT CAA TAT GAT GTA GTT TTC CAT GGG GTT TTA ACA GAT AAT GGG 182 Ly≥OVal Gin Tyr Asp Val Val Phe His Gly Val Leu Thr Asp Asn Gly 595 600 605

GAT ATT AGT AAC AAT AAG GCT CCA ATA GCA AAG GTA ACT GGA CCA AGC 187 Asp He Ser Asn Asn Lys Ala Pro He Ala Lys Val Thr Gly Pro Ser 610 615 620

ACT5GGT GCT GTA GGA AGA AAT ATT GAA TTT AGT GGA AAA GAT AGT AAA 192 Thr Gly Ala Val Gly Arg Asn He Glu Phe Ser Gly Lys Asp Ser Lys 625 630 635 640 GAT GAA GAT GGT AAA ATA GTA TCA TAT GAT TGG GAT TTT GGC GAT GGT 196 Asp Glu Asp Gly Lys He Val Ser Tyr Asp Trp Asp Phe Gly Asp Gly 645 650 655

GCA ACT AGT AGA GGC AAA AAT TCA GTA CAT GCT TAC AAA AAA GCA GGA 201 AlaSThr Ser Arg Gly Lys Asn Ser Val His Ala Tyr Lys Lys Ala Gly 660 665 670

ACA TAT AAT GTT ACA TTA AAA GTA ACT GAC GAT AAG GGT GCA ACA GCT 206 Thr Tyr Asn Val Thr Leu Lys Val Thr Asp Asp Lys Gly Ala Thr Ala 675 680 685

ACAOGAA AGC TTT ACT ATA GAA ATA AAG AAC GAA GAT ACA ACA ACA CCT 211 Thr Glu Ser Phe Thr He Glu He Lys Asn Glu Asp Thr Thr Thr Pro 690 695 700

ATA ACT AAA GAA ATG GAA CCT AAT GAT GAT ATA AAA GAG GCT AAT GGT 216 He Thr Lys Glu Met Glu Pro Asn Asp Asp He Lys Glu Ala Asn Gly 7055 710 715 720

CCA ATA GTT GAA GGT GTT ACT GTA AAA GGT GAT TTA AAT GGT TCT GAT 220 Pro He Val Glu Gly Val Thr Val Lys Gly Asp Leu Asn Gly Ser Asp 725 730 735

GAT GCT GAT ACC TTC TAT TTT GAT GTA AAA GAA GAT GGT GAT GTT ACA 225 AsfΛAla Asp Thr Phe Tyr Phe Asp Val Lys Glu Asp Gly Asp Val Thr 740 745 750

ATT GAA CTT CCT TAT TCA GGG TCA TCT AAT TTC ACA TGG TTA GTT TAT 230 He Glu Leu Pro Tyr Ser Gly Ser Ser Asn Phe Thr Trp Leu Val Tyr 755 760 765

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

TTT ATA TAT AAA CAC GAT TCT GCT TCA AAT ATA TCC TAT TCT TTA AAC 2448 Phe5He Tyr Lys His Asp Ser Ala Ser Asn He Ser Tyr Ser Leu Asn 805 810 815

ATA AAA GGA TTA GGT AAC GAG AAA TTG AAG GAA AAA GAA AAT AAT GAT 2496 He Lys Gly Leu Gly Asn Glu Lys Leu Lys Glu Lys Glu Asn Asn Asp 820 825 830

TCTOTCT GAT AAA GCT ACA GTT ATA CCA AAT TTC AAT ACC ACT ATG CAA 2544 Ser Ser Asp Lys Ala Thr Val He Pro Asn Phe Asn Thr Thr Met Gin 835 840 845

GGT TCA CTT TTA GGT GAT GAT TCA AGA GAT TAT TAT TCT TTT GAG GTT 2592 Gly Ser Leu Leu Gly Asp Asp Ser Arg Asp Tyr Tyr Ser Phe Glu Val 15850 855 860

AAG GAA GAA GGC GAA GTT AAT ATA GAA CTA GAT AAA AAG GAT GAA TTT 2640 Lys Glu Glu Gly Glu Val Asn He Glu Leu Asp Lys Lys Asp Glu Phe 865 870 875 880

GGT GTA ACA TGG ACA CTA CAT CCA GAG TCA AAT ATT AAT GAC AGA ATA 2688 Gl^OVal Thr Trp Thr Leu His Pro Glu Ser Asn He Asn Asp Arg He 885 890 895

ACT TAC GGA CAA GTT GAT GGT AAT AAG GTA TCT AAT AAA GTT AAA TTA 2736 Thr Tyr Gly Gin Val Asp Gly Asn Lys Val Ser Asn Lys Val Lys Leu 900 905 910

AGA5CCA GGA AAA TAT TAT CTA CTT GTT TAT AAA TAC TCA GGA TCA GGA 2784 Arg Pro Gly Lys Tyr Tyr Leu Leu Val Tyr Lys Tyr Ser Gly Ser Gly 915 920 925 AAC TAT GAG TTA AGG GTA AAT AAA TAATTTATC 281

Asn Tyr Glu Leu Arg Val Asn Lys 930 935

(2) INFORMATION FOR SEQ ID NO:2:

5 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 936 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Lys Gly He Glu Thr Phe Thr Glu Val Leu Arg Ala Gly Phe Tyr 1 5 10 15

Leu Gly Tyr Tyr Asn Asp Gly Leu Ser Tyr Leu Asn Asp Arg Asn Phe 20 25 30

GlhδAsp Lys Cys He Pro Ala Met He Ala He Gin Lys Asn Pro Asn 35 40 45

Phe Lys Leu Gly Thr Ala Val Gin Asp Glu Val He Thr Ser Leu Gly 50 55 60

Lys Leu He Gly Asn Ala Ser Ala Asn Ala Glu Val Val Asn Asn Cys 6Z0 70 75 80

Val Pro Val Leu Lys Gin Phe Arg Glu Asn Leu Asn Gin Tyr Ala Pro 85 90 95

Asp Tyr Val Lys Gly Thr Ala Val Asn Glu Leu He Lys Gly He Glu 100 105 110 Phe Asp Phe Ser Gly Ala Ala Tyr Glu Lys Asp Val Lys Thr Met Pro 115 120 125

Trp Tyr Gly Lys He Asp Pro Phe He Asn Glu Leu Lys Ala Leu Gly 130 135 140

LeuδTyr Gly Asn He Thr Ser Ala Thr Glu Trp Ala Ser Asp Val Gly 145 150 155 160

He Tyr Tyr Leu Ser Lys Phe Gly Leu Tyr Ser Thr Asn Arg Asn Asp 165 170 175

He Val Gin Ser Leu Glu Lys Ala Val Asp Met Tyr Lys Tyr Gly Lys 10 180 185 190

He Ala Phe Val Ala Met Glu Arg He Thr Trp Asp Tyr Asp Gly He 195 200 205

Gly Ser Asn Gly Lys Lys Val Asp His Asp Lys Phe Leu Asp Asp Ala 210 215 220

GI SLys His Tyr Leu Pro Lys Thr Tyr Thr Phe Asp Asn Gly Thr Phe 225 230 235 240

He He Arg Ala Gly Asp Lys Val Ser Glu Glu Lys He Lys Arg Leu 245 250 255

Tyr Trp Ala Ser Arg Glu Val Lys Ser Gin Phe His Arg Val Val Gly 20 260 265 270

Asn Asp Lys Ala Leu Glu Val Gly Asn Ala Asp Asp Val Leu Thr Met 275 280 285

Lys He Phe Asn Ser Pro Glu Glu Tyr Lys Phe Asn Thr Asn He Asn 290 295 300 Gly Val Ser Thr Asp Asn Gly Gly Leu Tyr He Glu Pro Arg Gly Thr 305 310 315 320

Phe Tyr Thr Tyr Glu Arg Thr Pro Gin Gin Ser He Phe Ser Leu Glu 325 330 335

GluδLeu Phe Arg His Glu Tyr Thr His Tyr Leu Gin Ala Arg Tyr Leu 340 345 350

Val Asp Gly Leu Trp Gly Gin Gly Pro Phe Tyr Glu Lys Asn Arg Leu 355 360 365

Thr Trp Phe Asp Glu Gly Thr Ala Glu Phe Phe Ala Gly Ser Thr Arg 10370 375 380

Thr Ser Gly Val Leu Pro Arg Lys Ser He Leu Gly Tyr Leu Ala Lys 385 390 395 400

Asp Lys Val Asp His Arg Tyr Ser Leu Lys Lys Thr Leu Asn Ser Gly 405 410 415

TyrδAsp Asp Ser Asp Trp Met Phe Tyr Asn Tyr Gly Phe Ala Val Ala 420 425 430

His Tyr Leu Tyr Glu Lys Asp Met Pro Thr Phe He Lys Met Asn Lys 435 440 445

Ala He Leu Asn Thr Asp Val Lys Ser Tyr Asp Glu He He Lys Lys 20450 455 460

Leu Ser Asp Asp Ala Asn Lys Asn Thr Glu Tyr Gin Asn His He Gin 465 470 475 480

Glu Leu Val Asp Lys Tyr Gin Gly Ala Gly Leu Pro Leu Val Ser Asp 485 490 495 Asp Tyr Leu Lys Asp His Gly Tyr Lys Lys Ala Ser Glu Val Tyr Ser 500 505 510

Glu He Ser Lys Ala Ala Ser Leu Thr Asn Thr Ser Val Thr Ala Glu 515 520 525

LysδSer Gin Tyr Phe Asn Thr Phe Thr Leu Arg Gly Thr Tyr Thr Gly 530 535 540

Glu Thr Ser Lys Gly Glu Phe Lys Asp Trp Asp Glu Met Ser Lys Lys 545 550 555 560

Leu Asp Gly Thr Leu Glu Ser Leu Ala Lys Asn Ser Trp Ser Gly Tyr 10 565 570 575

Lys Thr Leu Thr Ala Tyr Phe Thr Asn Tyr Arg Val Thr Ser Asp Asn 580 585 590

Lys Val Gin Tyr Asp Val Val Phe His Gly Val Leu Thr Asp Asn Gly 595 600 605

AspSIle Ser Asn Asn Lys Ala Pro He Ala Lys Val Thr Gly Pro Ser 610 615 620

Thr Gly Ala Val Gly Arg Asn He Glu Phe Ser Gly Lys Asp Ser Lys 625 630 635 640

Asp Glu Asp Gly Lys He Val Ser Tyr Asp Trp Asp Phe Gly Asp Gly 20 645 650 655

Ala Thr Ser Arg Gly Lys Asn Ser Val His Ala Tyr Lys Lys Ala Gly 660 665 670

Thr Tyr Asn Val Thr Leu Lys Val Thr Asp Asp Lys Gly Ala Thr Ala 675 680 685 Thr Glu Ser Phe Thr He Glu He Lys Asn Glu Asp Thr Thr Thr Pro 690 695 700

He Thr Lys Glu Met Glu Pro Asn Asp Asp He Lys Glu Ala Asn Gly 705 710 715 720

Pro5He Val Glu Gly Val Thr Val Lys Gly Asp Leu Asn Gly Ser Asp 725 730 735

Asp Ala Asp Thr Phe Tyr Phe Asp Val Lys Glu Asp Gly Asp Val Thr 740 745 750

He Glu Leu Pro Tyr Ser Gly Ser Ser Asn Phe Thr Trp Leu Val Tyr 10 755 760 765

Lys Glu Gly Asp Asp Gin Asn His He Ala Ser Gly He Asp Lys Asn 770 775 780

Asn Ser Lys Val Gly Thr Phe Lys Ala Thr Lys Gly Arg His Tyr Val 785 790 795 800

Pheδlle Tyr Lys His Asp Ser Ala Ser Asn He Ser Tyr Ser Leu Asn 805 810 815

He Lys Gly Leu Gly Asn Glu Lys Leu Lys Glu Lys Glu Asn Asn Asp 820 825 830

Ser Ser Asp Lys Ala Thr Val He Pro Asn Phe Asn Thr Thr Met Gin 20 835 840 845

Gly Ser Leu Leu Gly Asp Asp Ser Arg Asp Tyr Tyr Ser Phe Glu Val 850 855 860

Lys Glu Glu Gly Glu Val Asn He Glu Leu Asp Lys Lys Asp Glu Phe 865 870 875 880 Gly Val Thr Trp Thr Leu His Pro Glu Ser Asn He Asn Asp Arg He 885 890 895

Thr Tyr Gly Gin Val Asp Gly Asn Lys Val Ser Asn Lys Val Lys Leu 900 905 910

Arg5Pro Gly Lys Tyr Tyr Leu Leu Val Tyr Lys Tyr Ser Gly Ser Gly 915 920 925

Asn Tyr Glu Leu Arg Val Asn Lys 930 935

(2) INFORMATION FOR SEQ ID NO:3:

10 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 80 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

15 (ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

AAATAATTTA TCTTATAAAA AAGAGTGTGC CTAATACATG GCACACTCTT TTTATTTATT 6

TTTTTTCTTT TAAAAGATCC 8

International Application No: PCT/ /

MICROORGANISMS

Optional Sheet in connection with the microorganism referred to on page _2J , line 14 of the description:¹

A. IDENTIFICATION OF DEPOSIT:²

Further deposits ere identified on an additional sheet π

Name of Depository Institution⁶ American Type Culture Collection (ATCC)

Address of depository Institution (including postal code and country):⁴ 12301 Parklawn Drive Rockville, MD 20852 United States of America

Date of Deposit:⁶ Accession Number: June 22, 1993

B. ADDITIONAL INDICATIONS:⁷ (leave blank if not applicable). This information is continued on a separate attached sheet D

C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE:⁸ (If the indications are not for all designated States)

D. SEPARATE FURNISHING OF INDICATIONS:⁹ (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later. (Specify the general nature of the indications e.g., "Accession Number of Deposit")

Accession Number of Deposit

E. D This sheet was received with the International

□ The date of receipt (from the applicant) by the International Bureau:¹⁰

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Claims (26)

CLAIMS What is claimed is:

1. A recombinant DNA segment comprising DNA derived from Clostridium histolyticum coding for a polypeptide having the enzymatic activity of C. histolyticum collagenase, the polypeptide being distinguisable from the products of translation determined by the native expression of the C. histolyticum genomic coding sequence by having a higher molecular weight than the products of translation determined by the native expression of the C. histolyticum genomic coding sequence.

2. The DNA segment of claim 1 capable of being transcribed to yield mRNA, the mRNA capable of being translated to yield the polypeptide of claim 1.

3. The DNA segment of claim 1 capable of being transcribed to yield mRNA, the mRNA being capable of being translated to yield the polypeptide of claim 1 displaying the antigenicity of C. histolyticum collagenase.

4. The DNA segment of claim 1 wherein said DNA segment comprises a promoter derived from C. histolyticum such that said DNA segment when contained in the genome of a host cell can be transcribed under the control of said promoter and without the functioning of a promoter external to said DNA segment.

5. The DNA segment of claim 1 wherein the C. histolyticum structural gene is capable of further expressing polypeptides having collagenase activity, said polypeptides having molecular weights lower than the polypeptide of claim 1.

6. A vector comprising the DNA segment of claim 1 incorporated into a plasmid capable of transforming host cells, said plasmid selected from the group of plasmids consisting of those with both a drug resistance marker and a replication of origin.

7. The vector of claim 6 wherein said host cell is selected from the group consisting of E. coli. Bacillus subtilis, Clostridium histolyticum. and yeast.

8. E. coli host cells transformed with the vector of claim 6 and producing a polypeptide having collagenase activity and having the antigenicity of C. histolyticum collagenase, said polypeptide being distinguisable from the products of translation determined by the native expression of the C. histolyticum genomic coding sequence by having a higher molecular weight than the products of translation determined by the native expression of the C. histolyticum genomic coding sequence.

9« E. coli host cells transformed with the vector of claim 6 and producing polypeptides having collagenase activity wherein greater than about 50% by weight of the total polypeptides produced which have collagenase activity is comprised of a polypeptide having a higher molecular weight than the products of translation determined by the native expression of the C. histolyticum genomic coding sequence.

10. E. coli host cells transformed with the vector of claim 6 and producing a polypeptide having a molecular weight of about 110,000 daltons having collagenase activity, wherein said polypeptide of molecular weight of about 110,000 daltons comprises greater than about 50% by weight of the total polypeptides produced that have collagenase activity.

11. A substantially purified preparation of C. histolyticum collagenase, said collagenase having a higher molecular weight than the products of translation determined by the native expression of the C. histolyticum genomic coding sequence.

12. The preparation of claim 10 purified from E. coli transformed with a vector comprising Clostridium DNA.

13. A substantially purified preparation of C. histolyticum collagenase, said collagenase having a molecular weight of about 110,000 daltons and prepared from E. coli transformed with a vector comprising Clostridium DNA.

14. A composition comprising a substantially purified preparation of C. histolyticum collagenase, said collagenase having a higher molecular weight than the products of translation determined by the native expression of the C. histolyticum genomic coding sequence.

15. A composition comprising a substantially purified preparation of C. histolyticum collagenase, said collagenase having a molecular weight of about 110,000 daltons and prepared from E. coli transformed with a vector containing Clostridium DNA.

16. A method for digesting connective tissue and releasing embedded cells without destroying cell membranes and other essential structures comprising the steps of:

(a) incubating tissue in a buffered solution comprising said substantially purified collagenase of claim

11 with shaking at about 25-39° C to release and disperse the embedded cells; and

(b) separating the disperesed cells from tissue debris.

17. The method of claim 16 wherein the step of separating the dispersed cells from tissue debris is performed by density gradient centrifugation.

18. The method of claim 16 using a substantially purified preparation of C. histolyticum collagenase, said collagenase having a molecular weight of about 110,000 daltons and prepared from E. coli transformed with a vector comprising Clostridium DNA.

19. A method for isolating dispersed pancreatic islets comprising the steps of:

(a) incubating pancreatic tissue in a buffered solution comprising the substantially purifed collagenase of claim 11 with shaking at about 25-39° C to release and disperse the pancreatic islets; and

(b) separating the dispersed pancreatic islets from tissue debris.

20. The method of claim 19 wherein the step of separating the dispersed pancreatic islets is performed by density gradient centrifugation.

21. The method of claim 19 using a substantially purified preparation of C. histolyticum collagenase, said collagenase having a molecular weight of about 110,000 daltons and prepared from E. coli transformed with a vector comprising Clostridium DNA.

22. A method for dissociating tumors comprising the steps of:

(a) incubating tumor tissue in a buffered solution comprising the substantially purified collagenase of claim 11 with shaking at about 25-39° C to dissociate and disperse the tumor cells; and

(b) separating the dispersed tumor cells from tissue debris.

23. The method of claim 22 wherein the step of separating the dispersed tumor cells is performed by density gradient centrifugation.

24. The method of claim 22 using a substantially purified preparation of C. histolyticum collagenase, said collagenase having a molecular weight of about 110,000 daltons and prepared from E. coli transformed with a vector comprising Clostridium DNA.

25. A method for intradiscal treatment of herniation of nucleus pulposus comprising the steps of:

(a) preparing a sterile buffered solution comprising the substantially purifed collagenase of claim 11; and (b) injecting the sterile buffered solution comprising the substantially purified collagenase into the nucleus pulposus.

26. The method of claim 25 using a substantially purified preparation of C. histolyticum collagenase, said collagenase having a molecular weight of about 110,000 daltons and prepared from E. coli transformed with a vector comprising Clostridium DNA.