MXPA99004600A - Type i recombinant clostridium histolyticum collagenase and its use for isolating cells and cell aggregates - Google Patents

Abstract

A polypeptide having the properties of a Class I Clostridium histolyticum collagenase (CHC I) and the amino acid sequence SEQ ID NO:2, the N terminal of which has been optionally extended in one or more amino acids of the sequence SEQ ID NO:3, is advantageously adequate for isolating cells in tissues from mammals and human tissues.

Description

COLLAGENASE RECOMBINANT TYPE I OF CLOSTRIDIUM HISTOLYTICUM AND ITS USE TO INSULATE CELLS AND GROUPS OF CELLS FIELD OF THE INVENTION The invention concerns a type I recombinant collagenase (CCH I) from Clostridium his tolyticum and its use to isolate cells and groups of cells.

BACKGROUND OF THE INVENTION Bacterial collagenases, for example from Clostridium hi stolyticum, are used to digest tissues and to isolate individual cells or groups of cells (e.g., islets) (islets: Sutton et al., Transplantation 42 (1986) 689-691; Quibel et al., Anal. Bioc., 154 (1986) 26-28, bones: Hefley et al., J. Bone Mineral Res. 2 (1987) 505-516, umbilical cord: Holzinger et al., Immunol. 35 (1993) 109-118 Two different types of collagenase from Clostridium histolyticum are known (MF Frenen et al., J. Protein Chemistry 11 (1992) REF.: 30303 83-97) .The isolation and purification of collagenases from Clostridium histolyticum is described for example in EL Angleton and HE van Wart, Biochemistry 27 (1988) 7413-7418 and 7406-7412. Collagenases I were isolated and described by MD Bond and HE Van Wart, Biochemistry 23 (1984) 3077-3085 and 3085-3091 as well as by HE Van Wart, Biochemistry 24 (1985) 6520-6526. (molecular weight 68 D), ß (molecular weight 115 D),? (molecular weight 79 kD), and? (of molecular weight of 130 kD) are known as collagenases I. Collagenases I and collagenases II differ in their relative activity towards collagen and towards synthetic peptides. Collagenases I have greater activities towards collagen and gelatin and less activities towards short chain peptides than collagenase II (Bond and Wart, Biochemistry 23 (1984) 3085-3091). Additional collagenases are described in U.S. Patent Nos. 5,418,157 and 5,177,011 with a molecular weight of 68 kD. WO 91/14447 also describes a collagenase with a molecular weight of 68 kD. A recombinant collagenase with a molecular weight of approximately 110 kD is described in WO 94/00580 and a sequence is established therefor. Nothing is set forth in WO 94/00580 on the basis of its specificity and in particular whether it is a collagenase I or II. In addition to a collagenase with a molecular weight of 110 kD, a 125 kD collagenase is additionally mentioned which is also claimed to be recombinantly produced. However, WO 94/00580 does not give more details about this collagenase.

DESCRIPTION OF THE INVENTION The objective of the present invention is to provide a stable, highly active class I collagenase from Clostridium histolyticum (CCH I). The goal is achieved by a process for the production of a polypeptide which has the properties of a CCH I of the Clostridium histolyticum, has a given amino acid composition and can be obtained through the expression of an exogenous nucleic acid in prokaryotic or eukaryotic host cells and the isolation of the desired polypeptide wherein the nucleic acid encodes a polypeptide having the sequence of ID No.: 2 or a s1 polypeptide! e is N-terminally extended by one or several amino acids having the sequence of ID No .: 3. It has surprisingly turned out that a CCH I according to the invention has a high activity of collagenase class I and that it is very stable. Particularly preferred are a CCH I having the amino acid sequence SEQ ID NO: 2 and a CCH I that is N-terminally extended by one or more amino acids having SEQ ID NO: 3. CCH I in accordance with the invention it is a very pure enzyme that can be produced in large quantities. The CCH I according to the invention is not contaminated by other clos tridial enzymes and is free of toxins. The CCH I according to the invention is especially convenient for isolating cells from mammalian tissues, preferably from human tissue, for an application in cell therapy (transplant, immunotherapy) and for an application in gene therapy, in tissues: for example, pancreas, liver, bone, cartilage, skin, brain and nervous tissue, fat, muscle, heart, endothelium, kidney , solid tumors and for the purification of ulcers. In addition, this very pure enzyme (preferably in a mixture with other very pure enzymes (such as collagenase II and neutral protease)) is particularly suitable for the isolation of cells whose molecules / surface markers (antigens) should not be changed. Preferred applications thereof are, for example, the dissociation of solid tumors of all types, in vitro (for example, colon, breast, etc.) for adoptive immunotherapy and for general diagnosis, such as, for example, to detect rare cells of tissues and solid tumors, by markers / surface molecules, specific. The production of the recombinant CCH I can be carried out according to methods familiar to a person skilled in the art. For this, a DNA molecule is first produced which is capable of producing a protein having the activity of CCH I. The DNA sequence is cloned into a vector qv which can be transferred to a host cell and which can be replicated in the cell. same This vector contains promoter / operator elements that are necessary to express the DNA sequences in addition to the CCH I sequence. This vector that contains the sequence of CCH I and the promoter / operator elements is transferred to a host cell that is capable of expressing CCH I DNA. The host cell is cultured under conditions that are convenient for amplification of the vector and CCH I is isolated. In this process, adequate measures ensure that the protein can adopt an active tertiary structure in the cell. which exhibits the properties of CCH I. The nucleic acid sequence and the protein sequence can be modified to the usual degree. These modifications are, for example: - Modification of the nucleic acid to introduce several recognition sequences of the restriction enzymes, to facilitate the ligation, cloning and mutagenesis steps. - Modification of the nucleic acid to incorporate preferred codons for the host cell. -Extension of the nucleic acid by additional operator elements, to optimize expression in the host cell. Substitution or deletion of amino acids, while maintaining the basicity and spatial structure of the CCH I. It is advantageous to preserve 85% or more, and preferably 90% or more of the original amino acid sequence. A further subject of the invention is a polypeptide with the properties of a class I collagenase from Clostridium histolyticum with the amino acid sequence according to SEQ ID NO: 2 or a polypeptide extended N-terminally by one or more amino acids of SEQ ID NO: 3. A further subject of the invention is a nucleic acid encoding that protein.

The protein is preferably produced, recombinantly, in microorganisms, in particular in prokaryotes and in this case in E. coli. Suitable expression vectors must contain a promoter that allows the expression of the protein in the host organism. These promoters are known to a person skilled in the art and are, for example, the lac promoter (Chang et al., Nature 198 (1977) 1056), the trp promoter (Goeddel et al., Nuc.Aids Res. 1980) 4057), the? PL promoter (Shimatake et al., Nature 292 (1981) 128) and the T5 promoter (U.S. Patent No. 4,689,406). Synthetic promoters are also convenient, such as, for example, the tac promoter (U.S. Patent No. 4,551,433). Coupled promoter systems such as the T7-RNA polymerase / promoter system (Studier et al., J. Mol. Biol. 189 (1986) 113) are also convenient. Also suitable are hybrid promoters composed of a bacteriophage promoter and the operator region of the microorganism (EP-A 0 267 851). In addition to the promoter, an effective binding site to the ribosome is necessary. In the case of E. coli, this ribosome binding site is denoted as the Shine-Dalgarno (SD) sequence (Sambrook et al., "Expression of cloned genes in E. coli" in Molecular Cloning: A laboratory manual (1989). ) Cold Spring Harbor Laboratory Press New York, United States of America). To improve expression it is possible to express the protein as a fusion protein. In this case, a DNA sequence encoding the N-terminal part of an endogenous bacterial protein or for another stable protein is usually fused to the 5 'end of the DNA encoding CCH I.

Examples of this are, for example, lacZ (Phillips and Silhavy, Nature 344 (1990) 882- 884), trpE (Yansura, Meth. Enzymol. (1990) 161-166). After expression of the vector, preferably a biologically functional plasmid or a viral vector, the fusion proteins are preferably decomposed with enzymes (for example, factor Xa) (Nagai et al., Nature 309 (1984) 810). Other examples of cleavage sites are the cleavage site of the IgA protease (WO 91/11520, EP-A 0 495 398) and the cleavage site of ubiquitin (Miller et al., Bio / Technology 7 (1989) 698). ). The proteins expressed in this way, in bacteria, are isolated in the usual way by lysing the bacteria and isolating the protein. In a preferred embodiment it is possible to secrete the proteins as active proteins of the microorganisms. For this purpose, a fusion product is preferably used which is composed of a signal sequence which is suitable for the secretion of proteins in the host organisms used and the nucleic acid encoding the protein. In this process, the protein is either secreted into the medium (in the case of gram-positive bacteria) or into the periplasmic space (in the case of gram-negative bacteria). It is appropriate to place a cleavage site between the signal sequence and the sequence encoding the CCH I that allows the protein to be cleaved, either during processing or in an additional step. These signal sequences are derived, for example, from ompA (Ghrayeb et al., EMBO J. 3 (1984) 2437) or from pjoA (Oka et al., Proc. Nati, Acad. Sci. United States of America 82 (1985) 7212). In addition, the vectors also contain terminators. Terminators * are DNA sequences that signal the end of a transcription process. They are usually characterized by two structural characteristics: a region rich in repetitive G / C, inverse, which can form intramolecularly a double helix and a number of residues of U (or T). Examples are the main terminator in the DNA of the phage fd (Beck and Zink, Gene 16 (1981) 35-58) and rrnB (Brosius et al., J. Mol. Biol. 148 (1981) 107-127). In addition, expression vectors usually contain a selectable marker to select the transformed cells. Those selectable markers are, for example, the resistance genes for ampicillin, chloramphenicol, eri tromycin, kanamycin, neomycin and tetracycline (Davies et al., Ann.Rev. Microbiol., 32 (1978) 469). Selectable markers that are also convenient are genes for substances that are essential for the biosynthesis of substances necessary for the cell, such as for example histidine, tryptophan and leucine. Many suitable bacterial vectors are known. For example, vectors have been described for the following bacteria: Bacillus subtilis (Palva et al., Proc. Nati, Acad. Sci. USA 79 (1982) 5582), E. coli (Aman et al., Gene 40 (1985 ) 183; Studier et al., J. Mol. Biol. 189 (1986) 113), Streptococcus cremoris (Powell et al., Appl. Environ Microbiol. 54 (1988) 655), Streptococcus lividans and Streptomyces lividans (U.S. Patent No. 4, 747, 056). Additional genetic engineering methods for the construction and expression of suitable vectors are described in J. Sambrook et al., Molecular Cloning: A laboratory manual (1989), Cold Spring Harbor Laboratory Press, New York, N.Y. Apart from prokaryotic microorganisms, recombinant CCH I can also be expressed in eukaryotes (such as for example CHO cells, yeast or insect cells). The yeast system or insect cells is preferred as the eukaryotic expression system. Expression in yeast can be achieved by three types of yeast vectors (integration YIP vectors (yeast integration plasmids), replication vectors YRP (yeast replication plasmids) and episomal YEP vectors (yeast episomal plasmids). details of this are described, for example, in SM Kingsman et al., Tibtech 5 (1987) 53-57). The invention further concerns a process for disintegrating cell tissue and releasing cells or groups of cells contained therein, incubating the cell tissue with a class I collagenase from Clostridium histolyticum until the cells or groups of cells have been released to the desired degree and separating the cells or groups of cells from the fractions of the cell tissue. The separation of cells or groups of cells from the fractions of the cell tissue is preferably carried out by centrifugation, using a density gradient. Cells or groups of cells are usually isolated from tissues (eg, pancreas, liver, skin, endothelium, umbilical cord, bone, tumor tissue) by incubating the organs, parts of organs or tissues, with enzymes that dissolve the tissue matrix extracellular, surrounding connective (islets: Sutton et al., Transplantat ion 42 (1986) 689-691; Quibel et al., Anal. Biochem. 154 (1986) 26-28; bone: Hefley et al., J. Bone Mineral Res. 2 (1987) 505-516); umbilical cord: Holzinger et al., Im unol. Lett. 35 (1993) 109-118). Tumor cells isolated in this manner can be used advantageously for vaccination against tumors and / or for adoptive immunotherapy. Tissue disintegration can also be carried out by perfusing the entire organ (Ricordi et al., Diabetes 37 (1988) 413-420) with an enzyme solution. Important factors in this process, in addition to the composition of the enzymatic mixture, are the duration, the pH value and the temperature of the digestion, as well as the mechanical action, for example, by stirring and adding metal balls. Since the extracellular connective tissue matrix often has a high proportion of collagen, collagenases play a special role (Wolters, Hormone and Metabolic Research 26 (1994), 80). The process according to the invention is preferably used to isolate islets or islet cells, from pancreatic tissue. In addition, the addition of additional enzymes such as proteinases (e.g., neutral protease, see example 5 or other meto-protoplases;: serine proteases such as trypsin, chemo trypsin, plasmin, etc, cysteine proteases; Aspartate proteases), elastase, hyaluronidase, lipase or other collagenases, may be advantageous for the quality of digestion. The invention is further elucidated by the following examples, the figure and the sequence protocol. The sequence protocols denote: SEQ ID No. 1: DNA fragment of CCH I SEQ ID No. 2: Fragment of derived protein SEQ ID No. 3: N-terminal extension SEQ ID No. 4: 4-24: primers and peptide fragments Figure 1 shows the expression vector p-Lac-F-Coll -3 Example 1 Purification of class I collagenase (CC.H I) 1 g of collagenase P (from Clostridium histolyticum, Boehringer Mannheim GmbH, Catalog No. 1 213 857) was dissolved in 20 ml of H20 (Milli Q quality) and the particulate components were removed by centrifugation.

After precipitation with 60% ammonium sulfate, the precipitate was taken in 9.5 ml of H20 and dialyzed overnight at 4 ° C against 10 mM Tris-HCl, pH 7.5. The dialysed product was applied, at room temperature, to a chelate-Zn Sepharose column (volume of column VC = 50 ml, diameter 2 cm). Subsequently, the column was washed with 10 mM Tris-HCl, pH 7.5. The maximum eluent containing the CCH I fractions was further separated on a Mono Q column (Pharmacia) in Tris HCl, 10 mM / 1, pH 7.5, using a linear gradient up to 10 mM Tris / 1, pH 7.5, 0.3 NaCl M / l. The peak or maximum containing the activity of CCH I (the activity was determined as the azocoll activity according to Chavira et al., Anal. Biochem. 136 (1984) 446-450) is further purified by reverse phase HPLC ( mobile solvent A: TFA (trifluoroacetic acid / H20, 25% acetonitrile, step gradient up to 50% acetonitrile in 5 minutes and up to 70% acetonitrile in 15 minutes) The fraction of the second stage with gradients containing the maximum principal value , evaporated to dryness and, after carboxymethylation, is subjected to digestion with endoprot kinase Lys C. The peptides were also separated by reverse phase HPLC (Vydac C4) with a gradient of acetonitrile / 0.12% TFA, from 5 to 45%, in 105 minutes The peptides were pooled, evaporated to dryness and subjected to sequencing.

Example 2 Cloning of class I collagenase from Clostridium histolyticum 2. 1 A 1.4 kB segment of the CCH I gene that was derived from the peptide sequences that had been obtained by digestion of the purified collagenase I from Clostridium histolyticum was isolated with the aid of a PCR reaction in accordance with Example 1. This segment or fragments thereof was used to prepare a DNA probe that can be labeled, for example, with Dig. That probe is used to selectively classify gene banks to isolate the rest of the CCH I genes.

Peptide sequences: Coll-I-2Q: F N S P E E Y (SEQ ID NO: 4) Coll-I-72: E D G D V T I (SEQ ID NO: 5) Primers derived therefrom: Coll-l-2QF: 5 'tty a and wsn cen gar gar tay (SEQ ID No .: 6) Coll-l-2QR rt ytc ytc ngg nsw rtt raa (SEQ ID NO: 7) Coll -l-72F. gay gay gay gn gn acn att (SEQ ID NO: 8) Coll-l-72R. dat ngt nac rtc ncc rtc ytc (SEQ ID No .: 9) Four primers were designed and used in two different PCR reactions (combinations of 2QF / 72R and 2QR / 72F primers). Only the 2QF combination with 72R produced a PCR product (length of 1.4 kB) which corresponded to the sequence of position 2418 to 3806 of the total sequence in SEQ ID NO. : 1. The identity of this piece and the structure of reading on it were confirmed by the recovery of additional peptide sequences of Collagenase I from Clostridium histolyticum: Peptide 1-23: DWDEMSK (SEQ ID NO: 10) Peptide 1-33: VTGPSTGAVGRNIEFSGK (SEQ ID NO: 11) Peptide 1-40: NTEYQNHIQELVDK (SEQ ID NO: 12) 2.2 Selective Classification of the Gene Bank It was possible to isolate a 5.4 kB long DNA fragment containing the complete 1.4 kB probe from a gene bank that was prepared from genomic DNA of Clostridium histolyticum, divided with Stu I / HindII, with the aid of a labeled fragment. with digoxigenin (derived from the previous 1.4 kB fragment). DNA sequencing, from the ends of the known sequence of 1.4 kB, allowed the CCH I sequence to be completed at the 3f end and extended at the 5 'end to a division site Stu I. The sequence at the end 3 'corresponds to the position of 3807 to 4358 of the total sequence shown in SEQ ID NO. : 1. The sequence of an additional peptide of the wild-type enzyme was found in this section of sequences: Peptide 1-67: EDGDVTIELPYSGSSNFTWLVY (SEQ ID NO: 13) A large portion of this peptide overlaps with peptide 1-72 (SEQ ID NO: 5) but contains 4 additional amino acids that can not yet be found in the fragment of original DNA of 1.4 kB. The sequence at the 5 'end corresponds to the position of 1969 to 2417 of the total sequence shown in SEQ ID NO. : 1. It was also possible to identify a 4.6 kB DNA fragment from a Bgl II gene bank, using the labeled probe of 1.4 kB. This fragment proved not to be clonable as a whole, but it was possible to sequence it from the Stu I site forward and produced additional sequence information around the 5 'region. The sequence found corresponds to the position of 1630 to 1974 of the total sequence shown in SEQ ID NO. : 1. It was possible to confirm that this sequence belongs to the collagenase I gene, by the discovery of an additional peptide: peptide 1-42: QFRENLNQYAPDYVK (SEQ ID NO: 14) 2. 3 Reverse PCR In an experiment to obtain information about the 5 'region of the genes, by means of an inverse PCR, genomic DNA was separated from Clostridium histolyticum divided with Stul, in an agarose gel, the region from 1.9 to 2.7 kB was isolated (cut of the gel), it was re-ligated and amplified by reverse PCR: Used primers: 449-colll: 5'-aga caa tgc ett ggt atg g (SEQ ID No .: 15) 453-colll-R: 5'-gtg aaa gtt tet ata ecc ttc (SEQ ID No .: 16) A fragment of 1.85 kB was obtained that could not be cloned as a whole but it was possible to determine a partial sequence. This is the position of 1384 to the 1570 of the total sequence shown in SEQ ID NO: 1. This sequence is in the region of initiation and immediately upstream of the sequence previously described for collagenase I genes from Clostridium histolyticum. 2. 4 Cloning of the 5 * region of collagenase I genes from Clostridium histolyticum Genomic DNA from Clostridium histolyticum was digested with HindIII / Stul and separated on an agarose gel. The region was cut between 1.7 and 2.2 kB (the expected fragment in Southern spotting is 1.9 kB). The DNA was isolated from the gel by conventional methods (QiaEx) and cloned into pUC19 by HindIII / HindII. 2. 5 Selective classification of the gene bank: The HindIII / Stul gene bank was selectively screened with a Dig-labeled probe of approximately 400 bp in length. The sequence of the probe corresponds to the position of 1453 to 1852 of the total sequence shown in SEQ ID NO: 1. The probe was prepared with the aid of two primers: 464-colll: 5'-gta cag gtt etc aaa agt te (SEQ ID No .: 17) 448-colll-R: 5'-gtt cct tta acg taa tea gg (SEQ ID No .: 18) Among approximately 2,200 selectively classified clones, there were 14 with a positive signal, and the mini-preparations of the plasmid were? they were carried out in these using conventional methods, and the four best clones all contained the same insert corresponding to position 1-1971 of the total sequence shown in SEQ ID NO: 1. This sequence resulted in a total length for the genes of CCH I and its upstream region, of approximately 4.4 kB. The complete sequence is shown in SEQ ID NO. : 1. A large open reading structure is located in this sequence, which in the 3 'region is directed towards the already described part of collagenase I. Potential starting points, for translation, are located in the 1002 position ( ATG), 1029 (ATG), 1110 (ATG), as well as 1176 (GTG) and 1254 (GTG). 2. 6 Constructions extended in region 5 *: A N-terminus of a possible mature form of collagenase (IANTNSEK-SEQ ID NO: 19) determined by protein sequencing is found at position 1332 of SEQ ID NO: 1. The corresponding polypeptide is shown in SEC ID No .: 2. Since all the starting points, for the translation, described above, are also located at address 5? and remain within the structure, several expression constructs were prepared and their expression properties examined. Surprisingly it was found to start in several positions, each of which leads to actively expressed enzymes.

Examples of these constructs: A Smal or Aval cleavage site can be introduced in the 5 'position before ATG 1002, 1029, 1110 or GTG 1176 or 1254, through the proper selection of the primers. If an opposite primer is used that is located on the far side of the Brfl division site (1965-1970), then all others can be prepared from a pUCBM20 expression vector by exchange through Smal or Aval and the scintillation Brfl. starts in 1002: MTMITNSRA MKKNILKIL ... (SEQ ID NO: 20) starts in 1029: MTMITNSRA MDSYSKESK ... (SEQ ID No .: 21) starts in 1176: MTMITNSRA MKNIEEAKV ... (SEQ ID NO: 22 ) The underlined sequence is in each case a fusion part of the pUCBM20 resulting from the construction. It is known that GTG sequences can also be considered as a starting position. A particularly preferred start is found in GTG 1176 which can be converted to ATG by selecting a suitable primer: primer 472-Coll: 5 '-tcc cea att gee cgg gct atg aaa ata ata gaa gag gca aag (SEQ ID No .: 23) This leads to the expression vector placF-coll-3 (Fig. 1). 2. 7 Additional expression possibilities The expression constructs previously described use the lac promoter and the homologous signal peptide. Similarly, other promoters may be used instead of those, such as the Mgl (EP-B 0 285 152) or tac (EP-B 0 067 540). The signal peptide can also be replaced by others, for example, by the mgl signal peptide. That heterologous signal peptide could preferably be inserted directly opposite the mature end 5? of the collagenase gene. However, also in this case it was found that several positions can be conceived. Hence, an mgl signal in front of the IANTNSEK peptide sequence (SEQ ID NO: 19) and in front of AYLTMNTS (SEQ ID NO: 24) leads to the active expression of CCH I. The fusion part of pUCBM20 caused for the construction, it is not necessary either. The expression still takes place when the fusion part is shortened or totally eliminated. It is also possible to completely eliminate the signal and express the CCH I toplasmically. 2. 8 Strains: Preferentially, known strains of E. coli K12 such as C600, HB101, and simlares are used to express CCH I. Other prokaryotic and also gram positive systems (eg, bacillus or streptomyces) and expression can also be conceived. in eukaryotic cell systems. 2. 9 Molecular weight: purified (wild-type): approximately 115 kDa (after the electrophores is on SDS gel). purified (rec): approximately 115 kDa (after electrophoresis in SDS gel). theoretical. (IANTNSEK): 113897 theoretical. (AYLTMNTSS): 122547 Example 3 Expression of collagenase class I Fermentation of 10 1 of the expression clone of F. coli for CCH I and high pressure lysis. Pre-cultures are prepared from standard cultures (smears on plates, ampules or sequins) that are incubated at a temperature of 30 to 37 ° C while stirring. The volume of inoculation for the next highest dimension is 1 to 10% by volume, in each case. Ampicillin (50 to 100 mg / 1) is added to the pre-culture and the main culture, for selection against the loss of the plasmid. As nutrients enzymatically digested protein and / or yeast extract can be used, as a source of N and C, as well as glycerol and / or glucose, as an additional source of C. The medium is buffered to pH 7 and can be added metal salts at physiologically tolerated concentrations, to stabilize the fermentation process.

The fermentation is carried out as a batch of feed with a dosage of protein / yeast extract, mixed. The rate of growth is regulated by the proportion of the dosage and the pH value is controlled by glycerol and / or glucose as an acidity regulator. The partial pressure of the dissolved oxygen (p02) is maintained at a value greater than 20% to avoid anaerobic conditions, through the proportion of aeration, regulation of the rpm and proportion of the dosage. The temperature of the fermentation is 25 to 37 ° C. The growth is determined by measuring the optical density at 528 nm. The expression of CCH I is induced by IPTG or lactose. After a fermentation period of approximately 30 to 40 hours, the biomass is collected at the maintained optical density, by centrifugation. The biomass is taken in a suitable buffer and smoothed by a continuous, high pressure press at 1,000 bar. The suspension obtained in this way is centrifuged again and the supernatant containing the dissolved CCH I is further processed Example 4 Isolation of islets of porcine pancreas The pancreas is prepared from a freshly slaughtered pig and cooled in an ice-cold HBSS buffer (Gibco) until further processed. A cannula A is inserted into the pancreatic duct and fixed to it, the tightness of the pancreas, to the water, is analyzed using HBSS buffer. An enzyme solution was injected with HBSS + Ca2 + buffer containing recombinant, purified type I collagenase, alone, or in combination with a purified native or neutral recombinant protease, and / or type II collagenase from Clostridium histolyticum. The pancreas treated in this manner is connected to the perfusion unit which also contains the enzyme solution mentioned above (discontinuous perfusion). The digestion is carried out at a temperature that is between 4 ° C and 37 ° C for a period of 5 to 120 minutes. The pump stops after it is assumed that the time is optimal and the container containing the pancreas is shaken manually, carefully, for a time of 3 to 20 minutes. If necessary, the prior addition of metal balls further facilitates the mechanical dissociation of the tissue and the release of the islets from the surrounding exocrine tissue. Digestion is stopped by the addition of HBSS / 10% STF (fetal calf serum) and the suspension is filtered through a sieve (mesh size) 300 μm) to separate the coarse particles. The islets present in the filtrate are centrifuged for 10 minutes at 100 g in 250 ml Nalgene round bottom flasks. The supernatant is decanted and the pelotiform mass containing the islets is resuspended in 50 ml of STF. The islets can be further purified through a density gradient performed manually. Firstly, 7 ml of islet suspension are added to 250 ml round bottom Nalgene flasks. These are first covered with 93 ml of Ficoll ™ solution (f = 1.077 g / cm3), and then with 50 mi of the medium (RPMI 1640). These gradients are centrifuged for 10 minutes at 100 g in an oscillating rotor. Fractions of 10 ml are collected and the size, purity and performance of the islets stained with dithiozone are determined microscopically in each fraction or with the aid of image analysis.

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USA 79 (1982) 5582 Phillips and Silhavy, Nature 344 (1990) 882-884 Powell et al., Appl. Environ. Microbiol. 54 (1988) 655 Quibel et al, Anal. Biochem. 154 (1986) 26-28 Ricordi et al, Diabetes 37 (1988) 413-420 Sambrook et al., "Expression of cloned genes in E. coli" in Molecular Cloning: A laboratory manual (1989) Cold Spring Harbor Laboratory Press, New York, USA Shimatake et al., Nature 292 (1981) 128 Studier et al., J. Mol. Biol. 189 (1986) 113 Sutton et al., Transplantation 42 (1986) 689-691 U.S. Patent No. 4,551,433 U.S. Patent No. 4,689,406 U.S. Patent No. 4,747,056 U.S. Patent No. 5,177,011 U.S. Patent No. 5,418,157 Van Wart, HE , Biochemistry 24 (1985) 6520-6526 WO 91/11520 WO 91/14447 WO 94/00580 Wolters, Hormone and Metabolic Research 26 (1994) p. 80 Yansura, Meth. Enzymol. 185 (1990) 161-166 SEQUENCE PROTOCOL (1) GENERAL INFORMATION (i) APPLICANT: (A) NAME: BOEHRINGER MANNHEIM GMBH (B) STREET: Sandhofer Str. 116 (C) CITY: Manheim (E) COUNTRY: Germany (F) POSTAL CODE (ZIP) : D-68305 (G) TELEPHONE: 08856 / 60-3446 (H) TELEFAX: 08856 / 60-3451 (ii) ITEM OF THE INVENTION: RECOMBINANT COLLAGENASE TYPE I OF THE CLOSTRIDIUM HISTOLYTICUM AND ITS USE TO INSULATE CELLS AND CELL GROUPS (iii) NUMBER OF SEQUENCES: 24 (iv) COMPUTER READABLE FORM: (A) TYPE OF MEDIA: Flexible Disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30B (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 4358 base pairs (B) TYPE: nucleotides (C) HEBRAS: double strand (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: genomic DNA (ix) CHARACTERISTICS: (A) NAME / KEY: CDS "(LOCATION): 1332 ... 4358 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No .: 1: AAGCTTTTAT AATTCTCACT ATACTCTCTA GATGGtATAA TAAGAGCCA AAATACGAAG 60 AATATAATAA AATATAATTT AAAAAGGATG ATTTCAATGC GAAAAAGAGT TTTTTTGAAT 120 ATATTTTTTA TTTTATGTTC CTCAATTTTT TTTATGTCCT GCACAGGAAA ATTTCAGGTT 180 ATAGATCSGG GTGATGGGGG AGATGAAATT TATTTAAACA AACAAGATGG TGTGAGTTTT 240 3AGATTCCTA AAGTGTGGGA TAAAAATTAT AAGATTATCA CTTCTAGAGA TAAAAGATAT 300 GGCAAAAAGT TAACTTTTAA AAAAAAGGAT AAGAAATGCA ACGTTATACT TTTAGAAATA 360 TGGATTTTGA ATGAGGAATA TTGGAGTGAA TTTAAAGATG TTAGGAAGTT TAAACTTATA 420 GGTAAAAGCG AAAAAGGCGT AGTAGTTTAT TCAAGAGGTA AATTAGATAG CATATTAGAA 480 AATAATGGAT TGGACATTAT GCATCATAAA GAAGAGAAAA AGAAAGATAT AGAAAAAATG 540 TACATTAAAG ATGAAGAAAT TAGCGATAGA ATCAAAATAA TTAGAAATTA ATAAAñAAAT 600 GAAAATAGAft. AAATTCATTT TACTAAAAAT TTATGTTTAC TTTCTATAAC AATCTTTGTA 660 AACTGTAAAT ACTAATGTAG TATTTTTTAG AAAATAATAA TCTGTTAAAA AGTGTATTTA 720 GGAACTAAAA ATGAATAAAT TTATAAAAAC TATTTACAAT ATCTAAAATA ATGTATATAA 780 TTTTTTATTAA ATAGATTATT TTGGTATTAA GGGGGTGGTT GAAAGAATAA ACAGAAAATT 8 0 GATATAATTC AATAAATAAA ATCTAAAGAG AAATATCTAA GTAATACACA AATCTAATAT 900 TAAAAtCATT TTAATATTAA GAATATTTTG TTAATAGGTA AAGGTTAAAA GGCATTCTAT 960 TATTAAGGTT AAAAGGTATT AATTATTAAG GGGGATTATC TATGAAAAAA AATATTTTAA 1020 AGATTCTTAT GGATAGTTAT TCTAAAGAAT CTAAAATTCA AACTGTACGT AGGGTTACGA 1080 GTGTATCACT TTTAGCGGCA TATCT ACT TGAATACTTC AAGTTTAGTT TTAGCAAAAC 11 0 CAATAGAAAA TACTAATGAT ACTAGTATAA AAAATGTGGA GAAATTAAGA AATGCTCCAA 1200 AIGAAGAGAA TAGTAAAAAG GTAGAAGATA GTAAAAATGA TAAGGTAGAA CATGTGAAAA 1260 ATATAGAAGA GGCAAAGGTT GAGCAAGTTG CACCCGAAGT AAAATCTAAA TCAACTTTAA 1320 GAAGTGCTTC T ATA GCG AAT ACT AAT TCT GAG AAA TAT GAT TTT GAG TAT 1370 He Ala Asn Thr Asn Ser Glu Lys Tyr Asp Phe Glu Tyr 1 5 10 TTA AAT GGT TTG AGC TAT ACT GAA CTT ACA AAT TTA ATT AAA AAT ATA 1418 Leu Asn Gly Leu Ser Tyr Thr Glu Leu Thr Asn Leu lie Lys Asn lie 15 20 25 AAG TGG AAT CA ATT AAT GGT TTA TTT AAT TAT AGT ACA GGT TCT CAA 1466 Lys Trp Asn Gln lie Asn Gly Leu Phe Asn Tyr Ser Thr Gly Ser Gln 30 35 40 45 AAG TTC TTT GGA GAT AAA AAT CGT GTA CA GCT ATA ATT AAT GCT TTA 1514 Lys Phe Phe Gly Asp Lys Asn Arg Val Gln Ala lie He Asn Ala Leu 50 55 SO CAÁ GAA AGT GGA AGA ACT TAC ACT GCA AAT GAT ATG AAG GGT ATA GAA 1562 Gln Glu Ser Gly Arg Thr Tyr Thr Wing Asn Asp Met Lys Gly lie Glu 65 70 75 ACT TTC ACT GAG GTT TTA AGA GTr GGT TTT TAT TTA GGG TAC TAT AAT 1610 Thr Phe Thr Glu Val Leu Arg Wing Gly Phe Tyr Leu Gly Tyr Tyr Asn 80 85 90 GAT GGT TTA TCT TAT TAT AAT GAT AGA AAC TTC CAA GAT AAA TGT ATA 165T Asp Gly Leu Ser Tyr Leu Asn Asp Arg Asn Phe ßln Asp Lys Cys He 95 100 105 CCT GCA ATG ATT GCA ATT CAA AAA AAT CCT AAC TTT AAG CTA GGA ACT 1706 Pro Wing Met He Wing He Gln Lys Asn Pro Asn Phe Lys Leu Gly Thr 110 .115 120 125 GCA GTT CAA GAT GAA GTT ATA ACT TCT TTA GGA AAA CTA ATA GGA AAT 1754 Wing Val Gln Asp Glu Val He Thr Ser Leu Gly Lys Leu He Gly Asn 130 135 140 GCT TCT GCT AAT GCT GAA GTA GTT AAT AAT TGT GTA CCA GTT CTA AAA 1802 Wing Wing Wing Asn Wing Glu Val Val Asn Asn Cys Pro Val Leu Lys 145 150 155 CAA TTT AGA GAA AAC TAT AAT CAAT TAT GCT CCT GAT TAC GTT AAA GGA 1850 Gln Phe Arg Glu Asn Leu Asn Gln Tyr Ala Pro Asp Tyr Lys Gly 160 165 170 ACÁ GCT GTA AAT GAA TTA ATT AAA GGT ATT GAA TTC GAT TTT TCT GGT 898 Thr Wing Val Asn Glu Leu He Lys Gly He Glu Phe Asp Phe Ser Gly 175 1T0 185 GCT GCA TAT GAA AAA GAT GTT AAG ACÁ ATG CCT TGG TAT GGA AAA ATT 1946 Ala Ala Tyr Glu Lys Asp Val Lys Thr Met Pro Trp Tyr Gly Lys He 190 195 200 205 GAT CCA TTT ATA AAT GAA CTT AAG GCC TTA GGT CTA TAT GGA AAT ATA 1994 Asp Pro Phe He Asn Glu Leu Lys Ala Leu Gly Leu Tyr Gly- Asn He 210 215 220 ACÁ AGT GCA ACT GAS TGG GCA TCT GAT GTT GGA ATA TAC TAT TTA AGT 2042 Thr Ser Wing Thr Glu Trp Wing Ser Asp Val Gly He Tyr Tyr Leu Ser 223 230 235 AAA TTC GGG CTT TAC TCA ACT AAC CGA AAT GAC ATA GTA CAG TCA CTT 2090 Lys Phe Gly Leu Tyr Ser Thr Asn Arg Asn Asp He Val Gln Ser Leu 240 245 250 GAA AAG GCT GTA GAT ATG- TAT AAG TAT GGT AAA ATA GCC TTT GTA GCA 2138 Glu Lys Ala Val Asp Met Tyr Lys Tyr Gly Lys He Ala Phe Val Ala 255 260 265 ATG GAG AGA ATA ACT TGG GAT TAT GAT GGG ATT GGT TCT AAT GGT AAA 2186 Met Glu Arg He Thr Trp Asp Tyr Asp Gly He Gly Ser Asn Gly Lys 270 275 280 285 AAG GTG GAT CAC SAT AAG TTC TAT GAT GAT GAT GAA AAA CAT TAT CTG. 2234 Lys Val Asp His Asp Lys Phe Leu Asp Asp Wing G u Lys His Tyr Leu 290 295 300 CCA AAG ACAT TAT ACT TTT GAT AAT GGA ACC TTT ATT ATA AGA GCA GGG 2282 Pro Lys Thr Tyr Thr Phe Asp Asn Gly Thr Phe He He Arg Ala Gly 305 310 315 GAT AAG GTA TCC GAA GAA AAA ATA AAA AGG CTA TAT TGG GCA TCA AGA 2 »30 Asp Lys Val be Glu Glu Lys He Lys Arg Leu Tyr Trp Wing Ser Arg 320 325 330 GAA GTG AAG TCT CAÁ TTC CAT AGA GTA GTT GGC AAT GAT AAA GCT TTA 2378 Glu Val Lys Ser Gln Phe His Arg Val Val Gly Asn Asp Lys Ala Leu 335 340 345 GAG GTG GGA AAT GC. GT GAT GTT TTA ACT ATG AAA ATA TTT AAT AGC 2426 Glu Val Gly Asn Wing Asp Asp Val Leu Thr Met Lys He Phe Asn Ser 350 355 360 365 CCA GAA GAA TAT AAA TTT AAT ACC AAT ATA AAT GGT GTA AGT ACT GAT 2474 Pro Glu Glu Tyr Lys Phe Asn Thr Asn He Asn Gly Val Ser Thr Asp 370 375 330 AAT GGT GGT CTA TAT ATA GAA CCA AGA GGG ACT TTC TAC ACT TAT GAG 2522 Asn Gly Gly Leu Tyr He Glu Pro Arg Gly Thr Phe Tyr Thr Tyr Glu 385 390 395 AGA ACÁ CCT CAÁ CAÁ AGT ATA TTT AGT CTT GAA GAA TTG TTT AGA CAT 2570 Arg Thr Pro Gln Gln Ser He Phe Ser Leu Glu Glu Leu Phe Arg His 400 405 410 GAA TAT ACT CAC TAT CA GCG AGA TAT CTT GTA GAT GGT TTA TGG 2S18 Glu Tyr Thr His Tyr Leu Gln Ala Arg Tyr Leu Val Asp Gly Leu Trp 415 420 425 GGG CAA GGT CCA TTT TAT GAA AAA AAT AGA TTA ACT TGG TTT GAT GAA 2666 Gly Gln Gly Pro Phe Tyr Glu Lys Asn Arg Leu Thr Trp Phe Asp Glu 430 435 440 445 GGT ACÁ GCT GAA TTC TTT GCA GGA TCT ACC CGT ACA TCT GGT GTT TTA 2714 Gly Thr Wing Glu Phe Phe Wing Gly Ser Thr Arg Thr Ser Cly to Leu 450 455 460 CCA AGA AAA TCA ATA TTA GGA TAT TTG GCT AAG GAT AAA GTA CAT 2762 Pro Arg Lys Ser He Leu Gly Tyr Leu Wing Lys Asp Lys Val Asp His 465 470 475 AGA TAC TCA TAG AAG AAG ACT CTT AAT TCA GGG TAT GAT GAC AGT GAT 2810 Arg Tyr Ser Leu Lys Lys Thr Leu Asn Ser Gly Tyr Asp Asp Ser Asp 480 485 490 TGG ATG TTC TAT AAT TAT GGA TTT GCA GTT GCA CAT TAC CTA TAT GAA 2858 Trp Met Phe Tyr Asn Tyr Gly Phe Wing Val Wing His Tyr Leu Tyr Glu 495 500 505 AAA GAT ATG CCT ACA TTT ATT AAG ATG AAT AAA GCT ATA TTG AAT ACA 2906 Lys Asp Met Pro Thr Phe He Lys Met Asn Lys Ala He Leu Asn Thr 510 515 520 525 GAT GTG AAA TCT TAT GAT GAA ATA ATA AAA AAA TTA AGT GAT GAT GCA 2954 Asp Val Lys Ser Tyr Asp Glu He He Lys Lys Leu Ser Asp Asp Ala 530 535 540 AAT AAA AAT ACA GAA TAT .CAA AAC CAT ATT CAA GAG TTA GTA AAA 3002 Asn Lys Asn Thr Glü Tyr Gln Asn His Gln Glu Leu Val Asp Lys 545 5S0 555 TAT CA GGA GCT CTA CCT CTA GTA TAC GAT TAC TAT AAA GAT 3050 Tyr Gln Gly Wing Gly Leu Pro Leu Val Ser Asp Asp Tyr Leu Lys Asp 560 565 570 CAT GGA TAT AAG AAA GCA TCT GAA GTA TAT TCT GAA ATT TCA AAA GCT 3098 Q His Gly Tyr Lya Lys Wing Ser Glu Val Tyr Ser Glu He Ser Lys Wing 575 580 585 GCT TCT CTT ACÁ AAC ACT AGT GTA ACA GCA GAA AAA TCT CAA TAC TTT 3146 Wing being Leu Thr Asn Thr Ser Val Thr Wing Glu Lys Ser Gln Tyr Phe 590 595 600 605 5 AAC ACT TTC ACT TGA AGA GGA ACT TAT ACÁ GGT GAA ACT TCT AAA GGT 3194 Asn Thr Phe Thr Leu Arg Gly Thr Tyr Thr Gly Glu Thr Ser Lys Gly 610 615 620 GAA TTT AAA GAT TGG GAT GAA ATG AGT AAA AAA TTA GAT GGA ACT TTG 3242 Glu Phe Lys Asp Trp Asp Glu Met Ser Lys Lys Leu Asp Gly Thr Leu 0 625 630 635 GAG TCC CTT GCT AAA AAT TCT TGG AGT GGA TAC AAA ACC TTA ACA GCA 3290 Glu Ser Leu Wing Lys Asn Ser Trp Ser Gly Tyr Lys Thr Leu Thr Wing 640 645 650 5 TAC TTT ACG AAT TAT AGA GTT ACÁ AGC GAT AAT AAA GTT CAA TAT GAT 3338 Tyr Phe Thr Asn Tyr Arg Val Thr Ser Asp Asn Lys Val Gln Tyr Asp 655 660 665 GTA GTT TTC CAT GGG GTT TTA ACA GAT AAT GGG GAT ATT AGT AAC AAT 3386 Val Val Phe His Gly Val Leu Thr Asp Asn Gly Asp As As Asn 0 670 675 680 685 AAG GCT CCA ATA GCA AAG GTA ACT GGA CCA AGC ACT GGT GCT GTA GGA 3434 Lys Ala Pro He Ala Lys Val Thr Gly Pro Ser Thr Gly Ala Val Gl and 690 695 700 5 AGA AAT ATT GAA TTT AGT GGA AAA GAT AGT AAA GAT GAA GAT GGT AAA 3482 Arg Asn He Glu Phe Ser Gly Lys Asp Ser Lys Asp Glu Asp Gly Lys 705 710 715 ATA GTA TCA TAT GAT TGG GAT TTT GGC GAT GGT AGC AGT AGG GGC 3530 He Val Ser Tyr Asp Trp Asp Phe Gly Asp Gly Wing Thr Ser Arg Gly 720 725 730 0 AAA AAT TCA GTA CAT GCT TAC AAA AAA GCA GGA ACAT TAT AAT GTT ACÁ 357T Lys Aan Ser Val His Wing Tyr Lys Lys Wing Gly Thr Tyr Asn Val Thr 735 740 745 TTA AAA GTA ACT GAC GAT AAG GGT GCA ACE GCT ACA GAA AGC TTT ACT 3626 Leu Lys Val Thr Asp Asp Lys Gly Ala Thr Ala Thr Glu Ser Phß Thr 750 755 760 765 ATA GAA ATA AAG GAA GAT ACÁ ACÁ ACÁ CCT ATA ACT AAA GAA ATG 3674 He Glu He Lys Asn Glu Asp Thr Thr Thr Pro He Thr Lys Glu Met 770. 775 780 GAA CCT AAT GAT ATA AAA GAT GAG GCT AAT GGT CCA ATA GTT PAA GGT 3722 Glu Pro Asn Asp Asp He Lya Glu Wing Asn Gly Pro He Val < •;! "Gly 785 790 795 GTT ACT GTA AAA GGT GAT TTA AAT GGT TCT GAT GAT GCT GAT ACC TTC 3770 Go Thr Val Lys Gly Asp Leu Asn Gly Ser Asp Asp Wing Asp Thr Phe 800 805 810 TAT TTT GAT GTA AAA GAA GAT GGT GAT GTT ACA ATT GAA CTT CCT TAT 3818 Tyr Phe Asp Val Lys Glu Asp Gly Asp Val Thr He Glu Leu Pro Tyr 815 820 825 TCA GGG TCA TCT AAT TTC ATA TGG TTA GTT TAT AAA GAG GAG GAC GAT 3866 Ser Gly Ser Ser Asn Phe Thr Trp Leu Val Tyr Lys Glu Gly Asp Asp 830 835 840 845 CAÁ. AAT CAX ATT GCA AGT GGT ATA GAT AAG AAT AAC TCA AAA GTT GGA 3914 Gln Asn His He Wing Ser Gly He Asp Lys Asn Asn Ser Lys Val Gly 850 T5S 360 AC TTT AAA GCT AA AAA GGA AGA CAT TAT GTG TTT ATA TAT AAA CAC 3962 Thr Phe Lys Wing Thr Lys Gly Arg His Tyr Val Phe He Tyr Lys His 865 870 875 GAT TCT GCT TCA AAT ATA TCC TAT TCT TTA AAC ATA AAA GGA TTA GGT 4010 Asp Ser Ala Ser Asn He Ser Tyr Ser Leu Asn He Lys Gly Leu Gly 880 885 890 AAC GAG AAA TTG AAG GAA AAA GAA AAT AAT GAT TCT TCT GAT AAA GCT 4058 Asn Glu Lys Leu Lys Glu Lys Glu Asn Aan Asp Ser Ser A = p Lys Wing 895 900 905 ACÁ GTT ATA CCA AAT TTC AAT ACC ACT ATG CAA GGT TCA CTT TTA GGT 4106 Thr Val lie Pro Asn Phe Asn Thr Met Gln Gly Ser Leu Leu Gly 910 915 920 925 GAT GAT TCA AGA GAT TAT TAT TCT TTT GAG GTT AAG GAA GAA GGC GAA 4154 Asp Asp Ser Arg Asp Tyr Tyr Ser Phe Glu Val Lys Glu Glu Glu Glu 930 935 940 GTT AAT ATA GAA CTA GAT AAA AAG GAT GAA TTT GGT GTA ACTA TGG ACA 4202 Val Aan He Glu Leu Asp Lys Asp Glu Phe Gly Val Thr Trp Thr 945 S50 955 CTA CAT CCA GAG TCA AAT ATT AAT GAC AGA ATA ACT TAC GGA CA GTT 4250 Leu His Pro Glu Ser Asn He Asn Asp Arg He Thr Tyr Gly Gln Val 960 965 970 GAT GGT AAT AAG GTA TCT AAT AAA GTT AAA TTA AGA CCA GGA AAA TAT 4298 Asp Gly Asn Lys Val Ser Asn Lys Val Lys Leu Arg Pro Gly Lys Tyr 975 980 985 TAT CTA CTT GTT TAT AAA TAC TCA GGA TCA GGA AAC TAT GAG TTA AGG 4346 Tyr Leu Leu Val Tyr Lys Tyr Ser Gly Ser Gly Asn Tyr Glu Leu Arg 990 995 1000 1005 GTA AAT AAA TAA 4356 Val Asn Lys * (2) INFORMATION FOR SEQ ID NO. : 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1009 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein He Ala Asn Thr Asn Ser Glu Lys Tyr Asp Phe Glu Tyr Leu Asn Gly 1 5 10 15 Leu Ser Tyr Thr Glu Leu Thr Asn Leu He Lys Asn He Lys Trp Asn 20 25 30 Gln He Asn Gly Leu Phe Asn Tyr Ser Thr Gly Ser Gln Lys Phe Phe 35 40 45 Gly Asp Lys Asn Arg Val Gln Ala He He Asn Ala Leu Gln Glu Ser 50 55 60 Gly Arg Thr Tyr Thr Wing Asn Asp Met Lya Gly He Glu Thr Phe Thr 65 70 75 80 Glu Val Leu Arg Wing Gly Phß Tyr Leu Gly Tyr Tyr Asn Asp Gly Leu 85 90 95 Ser Tyr Leu Asn Asp Arg Asn Phe Gln Asp Lya Cys He Pro Wing Met 100 105 no He Wing He Gln Lys Asn Pro Asn Phe Lys Leu Gly Thr Wing Val Gln 115- 120 125 Asp Glu Val He Thr Ser Leu Gly Lys Leu He Gly Aan Wing Ser Wing 130 135 - 140 Asn Wing Glu Val Val Asn Asn Cys Val Pro Val Leu Lya Gln Phß Arg .145 150 155 160 Glu Asn Leu Asn Gln Tyr Ala Pro Asp Tyr Val Lys Gly Thr Wing Val 165 170 175 A = n Glu Leu He Lys Gly He Glu Phe Asp Phe Ser Gly Wing Wing Tyr 180, 185 190 Glu Lys Asp Val Lya "'hr Met Pro Trp Tyr Gly Lys He Aap Pro Phe 195 200 205 He Asn Glu Leu Lys Ala Leu Gly Leu Tyr Gly Asn He Thr Ser Wing 210 215 220 Thr Glu Trp Wing Being Asp Val Gly He Tyr Tyr Leu Ser Lys Phe Gly 225 230 235 240 Leu Tyr Ser Thr Asn Arg Asn Asp He Val Gln Ser Leu Glu Lys Wing 245 250 255 Val Asp Met Tyr Lys Tyr Gly Lys He Wing Phe Val Wing Met Glu Arg 260 265 270 He Thr Trp Asp Tyr Asp Gly He Gly Ser Asn Gly Lys Lys Val Asp 275 280 285 His Asp Lys Phe Leu Asp Asp Wing Glu Lys His Tyr Leu Pro Lys Thr 290 295 300 Tvr Thr Phe Asp Asn Gly Thr Phe He He Arg Wing Gly Asp Lys Val 305 310 315 320 Ser Glu Glu Lys He Lys Arg Leu Tyr Trp Wing Ser Arg Glu Val Lys 325 330 335 Ser Gln Phe His Arg Val Val Gly Asn Asp Lys Ala Leu Glu Val Gly 340 345 350 Asn Wing Asp Aap Val Leu Thr Met Lys He Phe Asn Ser Pro Glu Glu 355 360 365 Tyr Lys Phe As Asn As Asn Gly Val Ser Thr Asp Asn Gly Gly 370 375 380 Leu Tyr He Glu Pro Arg Gly Thr Phe Tyr thr Tyr Glu Arg Thr Pro 385 390 395 400 Gln Gln Ser He Phß Ser Leu Glu Glu Leu Phe Arg His Glu Tyr Thr 405 410 415 His. Tyr Leu Gln Wing Arg Tyr Leu Val Asp Gly Leu Trp Gly Gln Gly 420 425 430 Pro Phe Tyr Glu Lys Asn Arg Leu Thr Trp Phe Asp Glu Gly Thr Wing 435 440 445 Glu Phe Phe Wing Gly .Ser Thr Arg Thr Ser Gly Val Leu Pro Arg Lys 450 455 460 Ser He Leu Gly Tyr Leu Ala Lys Asp Lys Val Asp His Arg Tyr Ser 465 -.470 475 480 Leu Lys Lya Thr Leu Asn Ser Gly Tyr Asp Asp Ser Asp Trp Met Phe 485 490 495 Tyr Asn Tyr Gly Phe Wing Val Wing Hia Tyr Leu Tyr Glu Lya Asp Met 500 505 510 Pro Thr Phe He Lya Met Asn Lys Wing He Leu Asn Thr- Asp Val Lys 515 520 525 Ser Tyr Asp Glu He He Lys Lys Leu Ser Asp Asp Wing Asn Lys Asn 530 535 540 Thr Glu Tyr Gln Aan His He Gln Glu Leu Val Asp Lys Tyr Gln Gly 545 550 555 560 Wing Gly Leu Pro Leu Val Ser Asp Asp Tyr Leu Lys Asp His Gly Tyr 565 570 575 Lys Lys Ser Glu Val Tyr Ser Glu He Ser Lys Wing Wing Ser Leu 580 585 590 Thr Asn Thr Ser Val Thr Wing Glu Lys Ser Gln Tyr Phe Asn Thr Phe 595 600 605 Thr Leu Arg Gly Thr Tyr Thr Gly Glu Thr Ser Lys Gly Glu Phe Lys 610 615 620 Asp Trp Asp Glu Met Ser Lys Lys Leu Asp Gly Thr Leu Glu Ser Leu 625 630 635 640 Wing Lys Asn Ser Trp Ser Gly Tyr Lys Thr Leu Thr Wing Tyr Phß Thr € 45 650 655 Asn Tyr Arg Val Thr Ser Asp Asn Lys Val Gln Tyr Asp Val val Phe 660 665 670 His Gly Val Leu Thr Asp Asn Gly Asp He Ser Asn Asn Lys Ala Pro 675 680 685 He Ala Lys at Thr Gly Pro Ser Thr Gly Ala Val Gly Arg Asn He 690 695 700 Glu Phe Ser Gly Lya Asp Ser Lys Asp Glu Asp Gly Lys He Val Ser 705 710 715 720 Tyr Asp Trp Asp Phß Gly Asp Gly Wing Thr Ser Arg Gly Lys Asn Ser 725 730 735 Val His Wing Tyr Lys Lys Wing Gly Thr Tyr Asn Val Thr Leu Lys Val 740 745 750 Thr Asp Asp Lys Gly Wing Thr Wing Thr Glu Ser Phe Thr He Glu He 755 760 765 Lys Asn ßlu Asp Thr nr Thr Pro He Thr Lys Glu Met Glu Pro Asn 770 '775 780 Aap Asp He Lys Glu Wing Asn Gly Pro He Val Val Glu Val Val Val Valve 78S 790 795 800 Lys Gly Asp Leu Asn Gly Ser Asp Asp Wing Asp Thr Phe Tyr Phe Asp 805 810 815 Val Lys Glu Asp Gly Aap Val Thr He Glu Leu Pro Tyr Ser Gly Ser 820 825 830 Ser Asn Phe Thr Trp Leu Val Tyr Lys Glu Gly Asp Asp ßln Asn His 835 840 845 He Wing Ser Gly He Asp Lys Asn Asn Ser Lys Val Gly Thr Phe Lys 850 855 860 Wing Thr Lys Gly Arg Hia Tyr Val Phe He Tyr Lys His Asp Ser Wing 865 870 875 880 Being Asn He Being Tyr Being Leu Asn Ilß Lys Gly Leu 'Gly Asn- Glu Lys 885 890 895 Leu Lys slu Lys Glu Asn Asn Asp Ser Being Asp Lys Ala Thr Val He 900 905 910 Pro Asn Phe Asn Thr Met Gln Gly Ser Leu Leu Gly Asp Asp Ser 915 920 925 Arg Asp Tyr Tyr Ser Phe Glu Val Lys Glu Glu Gly Glu Val Asn He 930 935 940 Glu Leu Asp Lys Asp Glu Phß Gly Val Thr Trp Thr Leu His Pro 945 950 955 960 Glu Ser Asn He Asn Asp Arg He Thr Tyr Gly Gln val Asp Gly Asn 965 970 975 Lys Val Ser Asn Lys Val Lys Leu Arg Pro Gly Lys Tyr Tyr Leu Leu 980 985 990 Val Tyr Lys Tyr Ser Gly Ss? Gly Asn Tyr Glu Leu Arg Val Asn Lya 995 1000 1005 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 110 amino acids (B) TYPE: amino acid (C) HEBRAS: single strand (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: Met Lys Lys Asn He Leu Lya He Leu Met Asp Ser Tyr be Lys Glu 1 5 10 15 Be Lys Ha Gln Thr Val Arg Arg Val Thr Ser Val Ser Leu Leu Wing 20 25 30 Wing Tyr Leu Thr Met Asn Thr Ser Ser Leu Val Leu Wing Lys Pro He 35 40 45 Glu Asn Thr Asn Aap Thr Ser He Lya Asn Val Glu Lys Leu Arg Asn 50 55 60 Wing Pro Asn Glu Glu Asn Ser Lys Lys Val Glu Asp Ser Lys Asn Asp 65 70 75 80 Lys Val Glu His Val Lya Asn He Glu Glu Ala Lys Val Glu Gln Val 85 90 95 Wing Pro Glu Val Lys Ser Lys be Thr Leu Arg Ser Wing Ser 100 105 110 [2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) HEBRAS: single strand (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID No, Phe Asn Ser Pro Glu Glu Tyr 1 5 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) HEBRAS: single strand (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEC ID No .: í Glu Asp Gly Asp Val Thr He l 5 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) HEBRAS: single strand (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = primer (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No .: í TTYAAYWSNC CNGARGARTA Y INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) HEBRAS: single strand (D) TOPOLOGY: linear (ii) TYPE MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = primer (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO .: 1 RTAYTCYTCN GGNSWRTTRA A (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) HEBRAS: single strand (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = primer (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No .: 8 GARGAYGGNG AYG NACNAT T (2) INFORMATION FOR SEC ID No. 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) HEBRAS: single strand (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = primer (xi) DESCRIPTION OF SEQUENCE: SEQ ID No. DAT GTNACR TCNCCRTCYT C (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) HEBRAS: single strand (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No .: 10 Asp Trp Asp Glu Met Ser Lys 1 5 (2) INFORMATION FOR SEC ID No .: 11: (i) CHARACTERISTICS D? THE SEQUENCE: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) HEBRAS: single strand (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO .: eleven: Val Thr Gly Pro Sc.r Thr Gly Wing Val Gly Arg Asn lie Glu Phe 1 5 10 15 Ser Gly Lys (2) INFORMATION FOR SEC ID No .: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) HEBRAS: single strand (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. : 12: Asn Thr Glu Tyr Gln Asn His He Gln Glu Leu Val Asp Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) HEBRAS: single strand (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO .: 13: Glu Asp Gly Asp Val Thr He Glu Leu Pro Tyr Ser Gly Being 1 5 10 15 Asn Phe Thr Trp Leu Val Tyr 20 (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) 'LENGTH: 15 amino acids (B) TYPE: amino acid (C) HEBRAS: single strand (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO .: 14: Gln Phe Arg Glu Asn Leu Asn Gln Tyr Ala Pro Asp Tyr Val Lys 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 base pairs (B) TYPE: nucleotide (C) HEBRAS: single strand (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = primer (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO .: 15: AGACAATGCC TTGGTATGG (2) INFORMATION FOR SEC ID No .: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) HEBRAS: single strand (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = primer (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO .: 16: GTGAAAGTTT CTATACCCTT C (2) INFORMATION FOR SECTION ID No .: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleotide (C) HEBRAS: single strand (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION : / desc = primer (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO .: 17 GTACAGGTTC TCAAAAGTTC (2) INFORMATION FOR SEQ ID NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleotide (C) HEBRAS: single strand (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = primer (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 18 GTTCCTTTAA CGTAATCAGG (2) INFORMATION FOR SEQ ID NO, 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) HEBRAS: single strand (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO .: 19: He Ala Asn Thr Asn Ser Glu Lys 1 5 (2) INFORMATION FOR SEQ ID NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) HEBRAS: strand unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 20: Met Thr Met He Thr Asn Ser Arg Ala Met Lys Lys Asn He Leu 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) HEBRAS: single strand (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO .: 21: Met Thr Met He Thr Asn Ser Arg Ala Met Asp Ser Tyr Ser Lys 1 5 10 15 Glu Ser Lys (2) INFORMATION FOR SEQ ID NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) HEBRAS: single strand (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO .: 22: Met Thr Met He Thr Asn Ser Arg Ala Met Lys Asn He Glu Glu 1 5 10 15 Ala Lys Val (2) INFORMATION FOR SEC ID No .: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 42 base pairs (B) TYPE: nucleotide (C) HEBRAS: single strand (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = primer (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO .: 23: TTCCCAATTG CCCGGGCTAT GAAAAATATA GAAGAGGCAA AG (2) INFORMATION FOR SEQ ID NO: 24; (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) HEBRAS: single strand (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEC ID NO .: 24 Wing Tyr Leu Thr Met Asn Thr Ser 1 5 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. As described above, the invention is claimed as property contained in the following:

Claims (7)

1. The polypeptide characterized in that it has the properties of collagenase class I of Clostridium histolyticum (CCH I) and the amino acid sequence? SEQ ID NO: 2 which is optionally extended N-terminally by one or more amino acids of the sequence SEQ ID NO: 3.

2. The polypeptide according to claim 1, characterized in that for the same the nucleic acid encoding initiates nucleotide 1002, 1029, 1110, 1176 or 1254 of SEQ ID NO: 1.

3. The nucleic acid characterized in that it encodes a polypeptide with the properties of collagenase class I of Clostridium histolyticum (CCH I) having SEQ ID NO: 2 which is optionally extended in the N-terminus by one or more amino acids of the SEC ID No. : 34.

The nucleic acid according to claim 3, characterized in that it starts with nucleotide 1002, 1029, 1110, 1176 or 1254 of SEQ ID NO. : 1.

5. The process for the production of a polypeptide with the properties of class I collagenase from Clostridium histolyticum (CCH I) by the expression of an exogenous nucleic acid in prokaryotic or eukaryotic host cells and the isolation of the desired peptide, wherein the nucleic acid encodes for a polypeptide having SEQ ID NO: 2 or for a polypeptide that is extended N-terminally by one or more amino acids of the sequence SEQ ID NO: 3.

6. The process according to claim 5, characterized in that the nucleic acid starts with nucleotide 1002, 1029, 1110, 1176 or 1254 of SEQ ID NO: 1.

7. The use of a polypeptide according to claim 1 or 2, for the isolation of cells from a mammalian tissue and from a human tissue.