EP0799316A1 - Recombinant proteinase from clostridium hystolyticum and the use thereof for isolating cells and cell groups - Google Patents

Recombinant proteinase from clostridium hystolyticum and the use thereof for isolating cells and cell groups

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Publication number
EP0799316A1
EP0799316A1 EP95942186A EP95942186A EP0799316A1 EP 0799316 A1 EP0799316 A1 EP 0799316A1 EP 95942186 A EP95942186 A EP 95942186A EP 95942186 A EP95942186 A EP 95942186A EP 0799316 A1 EP0799316 A1 EP 0799316A1
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European Patent Office
Prior art keywords
dna
seq
nucleic acids
cell
nucleotides
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EP95942186A
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German (de)
French (fr)
Inventor
Friederike Hesse
Dorothee Ambrosius
Helmut Burtscher
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Roche Diagnostics GmbH
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Roche Diagnostics GmbH
Boehringer Mannheim GmbH
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Publication of EP0799316A1 publication Critical patent/EP0799316A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the invention relates to a recombinant proteinase (neutral protease, NP) from Clostridium histolyticum and its use for the isolation of cells and cell groups.
  • NP neutral protease
  • Proteolytic enzymes from Clostridium histolyticum are used to digest tissues and to obtain single cells or cell assemblies (e.g. islands) (islands: Sutton et al., Transplantation 42 (1986) 689-691; liver: Quibel et al., Anal. Bioche 154 (1986) 26-28; Bones: Hefley et al., J. Bone Mineral Res. 2 (1987) 505-516; Holzinger et al., Immunology Letters 35, 1993, 109-118).
  • Two different types of collagenase are known from Clostridium histolyticum (M.F. French et al., J. Protein Chemistry 11 (1992) 83-97).
  • NP neutral protease
  • Clostridium histolyticum a neutral protease from Clostridium histolyticum whose activity optimum is in the neutral pH range and which cleaves both casein and denatured collagen (Azocoll) (Mandl et al. , J. Clin. Invest 32, 1953, 1323-1329; Sparrow & McQuade, Biochim. Biophys. Acta 302, 1973, 90-94; Hefley, J. Bone Mineral Res. 2, 1987, 505-516).
  • This neutral protease is considered necessary as an auxiliary enzyme in the digestion of various tissues (bone: Hefley et al., Exp. Cell Res.
  • the NP has a molecular weight of approx. 35 kd (SDS gel electrophoresis).
  • the object of the present invention was therefore to provide nucleic acids which code for proteins with the activity of the neutral protease from Clostridium histolyticum, and to provide a process for their recombinant production.
  • the object is achieved by a nucleic acid which codes for a protein with the activity of the neutral protease from Clostridium histolyticum, characterized in that it is selected from the group comprising
  • nucleic acids mentioned in a) or b) would hybridize with one of the nucleic acids mentioned in a) or b).
  • nucleic acid of nucleotides 1027-1965 from SEQ ID NO: 3 is preferred. Also suitable are nucleic acids which are shortened or extended, for example by approximately 60 nucleotides, at the 5 'end compared to this nucleic acid. An extended nucleic acid of nucleotides 970-1965 which corresponds to a proform of the protease is particularly preferred. Shortened nucleic acids correspond to proteolytically, preferably autoproteolytically processed proteins.
  • Neutral protease cleaves both casein and denatured collagen.
  • Hybridization in the sense of the invention, is to be understood as a hybridization under the usual stringent conditions familiar to the person skilled in the art, as described, for example, by J. Sambrook in Molecular Cloning, Cold Spring Harbor Laboratory (1989) and B.D. Harnes, S.G. Higgins, Nucleic Acid Hybridization - A practical approach (1985), IRL-Press, Oxford, England. The standard protocols described in these publications are usually used for hybridization.
  • Stringent conditions is preferably understood to mean a hybridization in 6.0 x SSC at about 45 ° C. with a subsequent washing step at 2.0 x SSC at 50 ° C.
  • the salt concentration in the washing step can be selected, for example, from 2.0 x SSC at 50 ° C. for low stringency to 0.2 x SSC at 50 ° C. for high stringency.
  • the temperature of the washing step can be set between room temperature (22 ° C, low stringency) to about 65 ° C (high stringency).
  • the stringent hybridization Rung conditions are preferably chosen so that at least a homology of 75%, preferably 90%, is obtained in the amino acid sequence.
  • a DNA or RNA is suitable as nucleic acid in the sense of the invention.
  • the RNA is complementary to a DNA according to the invention.
  • the nucleic acid according to the invention can be of synthetic, semisynthetic or recombinant origin.
  • the peptide sequences NP23 (SEQ ID NO: 8, where Asp can also be in position 3 instead of Ala) and NP44 (SEQ ID NO: 4) were derived.
  • degenerate primers derived from these peptides a 300 bp fragment is obtained in the PCR reaction which does not code for a neutral protease.
  • a degenerate primer, derived from peptide NP44 alone an approximately 400 bp fragment is obtained which likewise does not code for the neutral protease.
  • the recombinant protease can be prepared by the methods familiar to the person skilled in the art.
  • a DNA is first produced, which is able to convert a protein produce, which has the activity of the protease.
  • Such a DNA which is selected from the group comprising
  • nucleic acids a) a DNA of nucleotides 1027-1965 from SEQ ID NO: 3 or a complementary DNA, b) nucleic acids that hybridize with a DNA of nucleotides 1027-1965 from SEQ ID NO: 3, c) nucleic acids that do not degenerate the genetic code, would hybridize with one of the nucleic acids mentioned in a) or b),
  • Such a vector contains promoter / operator elements which are required for the expression of the DNA.
  • This vector which contains the NP sequence and the promoter / operator elements, is transferred to a host strain that is capable of expressing the DNA of NP. The host cell is cultured under conditions suitable for amplifying the vector and NP is obtained. Suitable measures are taken to ensure that the protein can have an active tertiary structure in which it shows NP properties.
  • SEQ ID NO: 5 Proteins which contain essentially the same sequence and show similar properties are also suitable.
  • SEQ DD NO: 1 and SEQ ID NO: 2 show preferred DNA fragments.
  • a DNA of nucleotides 1027-1965 from SEQ ID NO: 3 is preferred.
  • An extended nucleic acid of nucleotides 970-1965 which corresponds to a proform of the protease is particularly preferred. Shortened nucleic acids correspond to proteolytically, preferably autoproteolytically processed proteins.
  • nucleic acid sequence of the protein can also be modified. Such modifications are, for example:
  • Another object of the invention is a method for producing a polypeptide, which has the properties of an NP from Clostridium histolyticum, by expressing an exogenous nucleic acid in prokaryotic or eukaryotic host cells and isolating the desired polypeptide, the DNA coding for said peptide is selected from the group comprising
  • nucleic acids mentioned in a) or b) would hybridize with one of the nucleic acids mentioned in a) or b).
  • a DNA of nucleotides 1027-1965 from SEQ ID NO: 3 is preferably used.
  • a biologically functional plasmid or a viral DNA vector which contains a nucleic acid according to the invention is used for expression. With this vector, a eukaryotic or prokaryotic host cell is stably transformed or transfected.
  • the protein is preferably expressed in microorganisms, in particular in prokaryotes, and there in E. coli.
  • the expression vectors must contain a promoter which allows expression of the protein in the host organism.
  • promoters are known to the person skilled in the art and are, for example, lac promoter (Chang et al., Nature 198 (1977) 1056), trp (Goeddel et al., Nuc. Acids Res. 8 (1980) 4057), ⁇ PL promoter (Shimatake et al., Nature 292 (1981) 128) and T5 promoter (U.S. Patent No. 4,689,406).
  • Synthetic promoters such as the tac promoter (US Pat. No. 4,551,433) are also suitable.
  • Coupled promoter systems such as, for example, T7 RNA polymerase promoter system (Studier et al., J. Mol. Biol. 189 (1986) 113) are also suitable.
  • Hybrid promoters comprising a bacteriophage promoter and the operator region of the microorganism (EP-A 0267 851) are also suitable.
  • An effective ribosome binding site is required in addition to the promoter.
  • E. coli referred to this ribosome binding site as the Shine-Dalgamo (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, USA).
  • a DNA which codes for the N-terminal part of an endogenous bacterial protein or another stable protein is usually attached to the 5 'end of the fused for the NP coding DNA.
  • Examples include lacZ (Phillips and Silhavy, Nature 344 (1990) 882-884), trpE (Yansura, Meth. Enzymol. 185 (1990) 161-166).
  • the fusion proteins obtained after expression are preferably cleaved with enzymes (e.g. factor Xa) (Nagai et al., Nature 309 (1984) 810). Further examples of the cleavage sites are the IgA protease cleavage site (WO 91/11520, EP-A 0 495 398) and the ubiquitin cleavage site (Miller et al., Bio / Technology 7 (1989) 698).
  • the proteins expressed in this way in bacteria are obtained in a conventional manner by digesting the bacteria and isolating the proteins.
  • a fusion product is preferably used, which consists of the signal sequence which is suitable for the secretion of proteins in the host organisms used and the nucleic acid which codes for the protein.
  • the protein is either secreted into the medium (for gram-positive bacteria) or into the periplasmic space (for gram-negative bacteria).
  • a cleavage site is expediently provided, which allows the protein to be split off either during processing or in an additional step.
  • signal sequences are, for example, ompA (Ghrayeb et al., EMBO J. 3 (1984) 2437), phoA (Oka et al., Proc. Natl. Acad. Sci. USA 82 (1985) 7212).
  • the vectors also contain terminators. Terminators are DNA sequences that signal the end of a transcription process. They are usually characterized by two structural peculiarities: an inverted repetitive G / C-rich region that can intramolecularly form a double helix, and a number of U (or T) residues. Examples are trp attenuator and terminator in the DNA of phage fd and rrnB (Brosius et al., J. Mol. Biol. 148 (1981) 107-127).
  • the expression vectors usually contain a selectable marker to select transformed cells.
  • selectable markers are, for example, the resistance genes for ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline (Davies et al., Ann. Rev. Microbiol. 32 (1978) 469).
  • suitable selectable markers are the genes for essential substances of the biosynthesis of substances necessary for the cell, such as histidine, tryptophan and leucine.
  • Suitable bacterial vectors are known.
  • vectors are described for the following bacteria: Bacillus subtilis (Palva et al., Proc. Natl. 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 ).
  • recombinant NPs can also be expressed in eukaryotes (such as CHO cells, yeast or insect cells).
  • eukaryotes such as CHO cells, yeast or insect cells.
  • the yeast system or insect cells is preferred as the eukaryotic expression system.
  • Expression in yeast can be via three types of yeast vectors (integrating YIp (yeast integrating plasmids) vectors, replicating YRp (yeast replicon plasmids) vectors and episomal YEp (yeast episomal plasmids) vectors.
  • yeast vectors integrating YIp (yeast integrating plasmids) vectors, replicating YRp (yeast replicon plasmids) vectors and episomal YEp (yeast episomal plasmids) vectors.
  • Another object of the invention is a method for dissolving cell tissue and releasing cells or cell assemblies contained therein by incubating the cell tissue with a neutral protease from Clostridium histolyticum, which is encoded by
  • This protease is free from other proteins from Clostridium histolyticum due to the recombinant production in a host cell which is different from Clostridium histolyticum.
  • the cells or cell assemblies are separated from the cell tissue portions preferably by centrifugation with a density gradient.
  • a protease is preferably used which is encoded by nucleotides 1027-1965 of SEQ ID NO: 3, or a form which is extended or shortened N-terminally by about 20 amino acids.
  • An extended protease which is encoded by a DNA of nucleotides 970-1965 from SEQ ED NO: 3 and which corresponds to a proform of the protease is particularly preferred.
  • Such an extended protease is degraded to a shortened form (e.g. protein with the amino acid sequence SEQ ID NO: 5) during transport through the membrane into the periplasm or into the medium by signal peptidases.
  • shortened N- or C-terminal forms which can arise, for example, autocatalytically.
  • Isolation of cells or cell groups from tissues is generally carried out by incubating organs, organ parts or tissues with enzymes that dissolve the surrounding extracellular connective tissue matrix (Islands: Sutton et al., Transplantation 42 (1986) 689-691; liver: Quibel et al., Anal. Biochem. 154 (1986) 26-28; bone: Hefley et al., J. Bone Mineral Res. 2 (1987) 505 - 516.
  • the proteinase according to the invention can also be used in the preparation of muscle cells (Maruyama et al “J. Pharmacol. Methods 19, 1988, 155-164), fat cells (Vendrell & Alemany, J. Biochem. Biophys. Methods 16, 1988, 49-54) , Ovarian and uterine tissue (Marcus et al., Endocrine Res. 10, 1984, 151-162), epithelial cells (Kaunitz, Am. J. Physiol. 254, 1988, 6502-6512), heart cells (Haworth et al., Cell Calcium 10, 1989, 57-62) and placenta tissue (Morrish & Siy, Endocrine Res. 12, 1986, 229-253) can be used.
  • Tissue disintegration can also be achieved by perfusing the entire organ (Ricordi et al, Diabetes 37 (1988) 413-420) with enzyme solution.
  • enzyme solution In addition to the composition of the enzyme mixture, the duration, the pH value and the temperature of the digestion, and also the mechanical action, for example by shaking and adding, are decisive Metal balls. Since extracellular connective tissue matrix often has a high proportion of collagen, the collagenases and the neutral protease play a special role. (Wolters, Hormone and Metabolie Research 26 (1994) p. 80)
  • the method according to the invention is preferably used for isolating islets or islet cells from pancreatic tissue.
  • Further preferred applications are the extraction of cells from tissues of all kinds for the establishment of cell cultures or for the extraction of cells which are used for gene or cell therapeutic purposes (cell engineering).
  • the proteinase according to the invention and the method according to the invention for dissociation can also be used ⁇ tion of tumor tissue, preferably ex vivo. After genetic modification, the tumor cells isolated in this way are e.g. returned to the patient to effect immunization against the tumor, for example for adoptive immunotherapy.
  • the plasmid pNP-86-IR / 23F was deposited on December 9, 1994 at the German Collection of Microorganisms and Cell Cultures GmbH (DSM), Mascheroder Weg 1b, D-38124 Braunschweig under the number DSM 9578.
  • the plasmid pUC21-E-NP which contains the bases 933-2100 of SEQ ID NO: 3, was released on November 23, 1995 at the German Collection of Microorganisms and Cell Cultures GmbH (DSM), Mascheroder Weg lb, D-38124 Braunschweig under the Number DSM 10341 deposited.
  • Fig. 1 shows a rough restriction cold of the neutral protease.
  • SEQ ID NO: 1 DNA fragment of the neutral protease
  • SEQ ID NO: 2 DNA fragment of the neutral protease
  • SEQ ID NO: 3 DNA of the neutral protease with flanking
  • SEQ ID NO: 4 8, 10, 13, 14 and 16 peptides for the derivation of primers SEQ ID NO: 6, 7, 9, 11, 12, 15, 17-20 primer sequences.
  • Lyophilisate of collagenase P (BM / Order No. 1213857) was dissolved in 5 mM HEPES, pH 7.5, 1 M CaCl 2 and centrifuged. The supernatant was pumped onto a Q-Sepharose column, which was equilibrated with the same buffer (loading: max. 20 mg Lyo / ml column material). After washing the column with the equilibration buffer until the baseline was reached, the NP was eluted with an increasing CaCl 2 gradient (1 - 150 mM, 10-fold). The fractions with high caseinolytic activity (resorufin-casein) were pooled. On the SDS gel there is a clear band at approx. 33 kD, visible impurities in addition to the "brown pigment" in the SDS gel are visible bands at approx. 50 kD (depending on the batch) and in the low molecular weight range ( ⁇ 10 kD) .
  • the NP fraction is drawn up at 4 ° C. on a butyl 650 C column (equilibrated with 10 mM Tris, pH 7.5, 5 mM CaCl 2 ). While the impurities (brown pigment, double band at approx. 50 kDa) are breakthrough, the NP is bound to the hydrophobic column material under these conditions. Elution was carried out at 4 ° C. with 10 mM Tris, pH 8.3, 5 mM CaCl 2 and 10% isopropanol or 30% isopropanol. The protein obtained in this way is homogeneous according to SDS-PAGE (> 95% clean).
  • This high-purity NP preparation (increase in the specific activity against resorufin-casein by a factor of 100) was digested with trypsin and the peptides separated on a reversed-phase HPLC column (C8). After the peptides had been concentrated to dryness, the amino acid sequence was determined.
  • the associated (degenerate) DNA sequence is derived from the peptide sequences determined after the purification of the neutral protease according to Example 1. Sequences that show an advantageous (low) denaturation are used, for example, to produce a labeled DNA sample via PCR for screening gene banks.
  • peptides NP 23 and NP 86 from which the following primers can be derived:
  • Peptide NP 23 (SEQ ID NO: 8)
  • Primer NP 23F (SEQ ID NO: 9)
  • Primer NP 23R (SEQ ID NO: 6)
  • Peptide NP 86 (SEQ ID NO: 10)
  • Primer NP 86-1F (SEQ ID NO: 11)
  • Primer NP 86-1R (SEQ ID NO: 12)
  • This approximately 320 bp long fragment can be labeled well with dig-dUTP, e.g. likewise in a PCR reaction, and serves as a probe for the identification of positive clones from a gene bank.
  • the gene bank can be produced in a generally known manner from Clostridium histolyticum DNA after digestion with restriction enzymes.
  • the approximately 320 bp long fragment was sequenced and contains DNA of the sequence SEQ ID NO: 2. This sequence is also contained in the plasmid pNP-86-IR / 23F, which was deposited with the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg lb, 38124 Braunschweig (DSM) under No. 9578 on December 9, 1994.
  • Clostridium histolyticum genomic DNA was digested with HindIII and the fragments were then ligated with DNA ligase. From the 320 bp fragment two outward-directed primers, NPC5 (SEQ ID NO: 5) and NPC6 (SEQ ID NO: 15), were derived and used in a PCR reaction. A 586 bp fragment (SEQ ID NO: 1) was obtained which contained the majority of SEQ ID NO: 2. However, no further pieces of the neutral protease gene could be found in this way.
  • the 586 bp fragment also contains the following further peptides from protein digestion that can be used to identify the reading frame:
  • NP-NT2 (SEQ ID NO: 13)
  • NP58 (SEQ ID NO: 14)
  • the PCR primer NP-19R (SEQ ID NO: 17) was derived from a new peptide, NP19 (SEQ ID NO: 16).
  • a PCR reaction with primer NP-19R and the primer 416 (SEQ ID NO: 18) derived from the 320 bp fragment resulted in a 488 bp fragment which contained further sequence information of the neutral protease (approx. 350 bp beyond SEQ ID 2) ). Since the reading frame 3 'was still open, another small piece of the gene was still missing.
  • a rough restriction map could already be created by mapping the area of the neutral protease gene with the help of Southern analyzes (Fig.
  • the genomic DNA from Clostridium histolyticum was cut with EcoRV and Seal, the fragments were separated in the agarose gel and analyzed in a Southern blot. Fragments in the size range around 1000 bp were isolated from the gel, since such a piece of DNA would have to contain the remaining 3 'sequence based on the restriction map. The DNA fragments isolated from the gel were ligated (blunt end).
  • the plasmid pUC21-E-NP which contains the bases 933-2100 of SEQ ID NO: 3, was released on November 23, 1995 at the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg lb, D-38124 Braunschweig, under the no. DSM 10341 deposited.
  • Example 3
  • DNA fragments which contain the gene for neutral protease or parts thereof are suitably modified at the ends (e.g. via PCR) and used as a whole or in combination in an expression vector for E. coli.
  • Preferred promoters are those which can be regulated well, e.g. lac, tac, trc, mgl; However, using other promoters is quite conceivable. Either the neutral protease's own signal peptide or a heterologous one (such as mgl, PhoA) can be used for secretion.
  • An E. coli strain that has been transformed with an expression plasmid is grown either in minimal medium or LB medium with antibiotic selection overnight at 30 or 37 ° C (e.g. in 5 ml culture).
  • the overnight culture is inoculated into a larger volume (e.g. 1 1) and allowed to continue growing. If this volume is already used for biomass production, e.g. when using a lac promoter at an OD-550 of 0.2 to 2, induce with IPTG and let the cells continue to grow until the OD and / or the enzyme activity formed no longer increases.
  • centrifugation is carried out and the biomass is used to purify neutral protease.
  • a considerable part of the neutral protease activity can also be found in the medium; then the medium is also collected and neutral protease is purified therefrom.
  • the pancreas is prepared from a freshly slaughtered pig and cooled in ice-cold HBSS buffer (Gibco) until further processing.
  • a Braunulse is inserted and fastened into the pancreatic duct, the tightness of the pancreas is checked with HBSS buffer.
  • An enzyme solution in HBSS buffer + Ca2 + which contains recombinant neutral protease from Clostridium histolyticum alone or in a mixture with purified collagenase type I or II, is injected.
  • the pancreas treated in this way is connected to the perfusion unit (discontinuous perfusion) which also contains the above enzyme solution.
  • the digestion takes place between 4 ° C and 37 ° C in a period of 5 to 120 minutes, the enzyme solution in the vessel being constantly pumped into the pancreas. After the time assumed to be optimal (usually 20 to 30 minutes until islands are released), the pump is stopped and the vessel containing the pancreas is carefully cauterized for 3 to 20 minutes Handshake. Metal balls added beforehand additionally facilitate the mechanical dissociation of the tissue and the release of the islands from the surrounding exocrine tissue. The course of the digestion is checked microscopically after dithizone staining of samples taken at regular intervals.
  • the digestion is stopped by adding ice-cold HBSS / 10% FCS (fetal calf serum) and the suspension is filtered through a sieve (mesh size 300 ⁇ m) in order to separate the large parts.
  • FCS fetal calf serum
  • the islands in the flow are centrifuged at 100 g in 250 ml Nalgene round-bottom bottles for 10 minutes. The supernatant is removed and the pellet containing the islets is resuspended in 50 ml FCS.
  • a further purification of the islands can be done using a density gradient made by hand.
  • ATAAAATCCT AAATGGTATG TTTGAAAATG GAAGATATTT TTTAGCAGAT TCCACCAGAC 180 CTTCAAATGG ATATATATTA ACATATGATG CTAATAACCA AGAGTATGGT TTCCCAGGTA 240

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Abstract

A process is proposed for breaking down cellular tissue and releasing the constituent cells or cell groups by incubating the cellular tissue with a recombinant neutral protease from Clostridium hystolyticum. The protease in question is coded by (a) a DNA of the nucleotide 1027-1965 from SEQ ID NO:3 or by a DNA complementary to it; (b) nucleic acids which hybridise with a DNA of the nucleotide 1027-1965 from SEQ ID NO:3; (c) nucleic acids which would hybridise with one of the nucleic acids mentioned in (a) or (b) without degeneration of the genetic code. The protease is a product of a prokaryotic or eukaryotic expression of an exogenic nucleic acid. Incubation continues until the release of cells or cell groups in the desired quantity and separation of the cells or cell groups from the cellular tissue fractions.

Description

Rekombinante Proteinase aus Clostridium histolyticum und ihre Verwendung zur Isolierung von Zellen und Zellverbänden Recombinant proteinase from Clostridium histolyticum and its use for the isolation of cells and cell assemblies
Gegenstand der Erfindung ist eine rekombinante Proteinase (neutrale Protease, NP) aus Clostridium histolyticum und ihre Verwendung zur Isolierung von Zellen und Zellverbänden.The invention relates to a recombinant proteinase (neutral protease, NP) from Clostridium histolyticum and its use for the isolation of cells and cell groups.
Proteolytische Enzyme aus Clostridium histolyticum, werden zum Verdau von Geweben und zur Gewinnung von Einzelzellen oder Zellverbänden (z.B. Inseln) verwendet (Inseln: Sutton et al., Transplantation 42 (1986) 689 - 691; Leber: Quibel et al., Anal. Bioche . 154 (1986) 26 - 28; Knochen: Hefley et al., J. Bone Mineral Res. 2 (1987) 505 - 516; Holzinger et al., Immunology Letters 35, 1993, 109 - 118). Aus Clostridium histolyticum sind zwei verschie¬ dene Collagenase-Typen bekannt (M.F. French et al., J. Protein Chemistry 11 (1992) 83 - 97).Proteolytic enzymes from Clostridium histolyticum are used to digest tissues and to obtain single cells or cell assemblies (e.g. islands) (islands: Sutton et al., Transplantation 42 (1986) 689-691; liver: Quibel et al., Anal. Bioche 154 (1986) 26-28; Bones: Hefley et al., J. Bone Mineral Res. 2 (1987) 505-516; Holzinger et al., Immunology Letters 35, 1993, 109-118). Two different types of collagenase are known from Clostridium histolyticum (M.F. French et al., J. Protein Chemistry 11 (1992) 83-97).
Neben verschiedenen Isoformen der Collagenase von Typ I und II ist auch eine neutrale Protease (NP) aus Clostridium histolyticum bekannt, deren Aktivitäts-Optimum im neutralen pH-Bereich liegt und die sowohl Casein als auch denaturiertes Collagen (Azocoll) spaltet (Mandl et al., J. Clin. Invest 32, 1953, 1323 - 1329; Sparrow & McQuade, Biochim. Biophys. Acta 302, 1973, 90 - 94; Hefley, J. Bone Mineral Res. 2, 1987, 505 - 516). Diese neutrale Protease wird als Hilfsenzym beim Verdau verschiedener Gewebe als notwendig erachtet (Knochen: Hefley et al., Exp. Cell Res. 149, 1983, 227 - 236; Pankreas: Wolters et al., Diabetologica 35, 1992, 735 - 742). Die NP hat ein Molekulargewicht von ca. 35 kd (SDS- Gelelektrophorese).In addition to various isoforms of type I and II collagenase, a neutral protease (NP) from Clostridium histolyticum is also known whose activity optimum is in the neutral pH range and which cleaves both casein and denatured collagen (Azocoll) (Mandl et al. , J. Clin. Invest 32, 1953, 1323-1329; Sparrow & McQuade, Biochim. Biophys. Acta 302, 1973, 90-94; Hefley, J. Bone Mineral Res. 2, 1987, 505-516). This neutral protease is considered necessary as an auxiliary enzyme in the digestion of various tissues (bone: Hefley et al., Exp. Cell Res. 149, 1983, 227-236; pancreas: Wolters et al., Diabetologica 35, 1992, 735-742) . The NP has a molecular weight of approx. 35 kd (SDS gel electrophoresis).
Um neutrale Protease zur Isolierung von Zellen und Zellverbänden in großem Umfang einset¬ zen zu können, ist es erforderlich, die neutrale Protease in reproduzierbarer Qualität und in großen Mengen zur Verfugung zu stellen. Dies ist durch ein rekombinantes Herstellverfahren möglich.In order to be able to use neutral protease for the isolation of cells and cell assemblies on a large scale, it is necessary to provide the neutral protease in reproducible quality and in large quantities. This is possible using a recombinant manufacturing process.
Die Aufgabe der vorliegenden Erfindung war es demzufolge, Nukleinsäuren, die für Proteine mit der Aktivität der neutralen Protease aus Clostridium histolyticum codieren, sowie ein Verfahren zu deren rekombinanter Herstellung zur Verfugung zu stellen. Die Aufgabe wird gelöst durch eine Nukleinsäure, welche für ein Protein mit der Aktivität der neutralen Protease aus Clostridium histolyticum codiert, dadurch gekennzeichnet, daß sie ausgewählt ist aus der Gruppe umfassendThe object of the present invention was therefore to provide nucleic acids which code for proteins with the activity of the neutral protease from Clostridium histolyticum, and to provide a process for their recombinant production. The object is achieved by a nucleic acid which codes for a protein with the activity of the neutral protease from Clostridium histolyticum, characterized in that it is selected from the group comprising
a) eine DNA der Nukleotide 1027-1965 aus SEQ ID NO:3 oder eine hierzu komplementäre DNA, b) Nukleinsäuren, die mit einer DNA der Nukleotide 1027-1965 aus SEQ FD NO: 3 hybridisieren, c) Nukleinsäuren, die ohne die Degeneration des genetischen Codes, mit einer der in a) oder b) genannten Nukleinsäuren hybridisieren würden.a) a DNA of nucleotides 1027-1965 from SEQ ID NO: 3 or a complementary DNA, b) nucleic acids that hybridize with a DNA of nucleotides 1027-1965 from SEQ FD NO: 3, c) nucleic acids that do not degenerate of the genetic code, would hybridize with one of the nucleic acids mentioned in a) or b).
Bevorzugt ist eine Nukleinsäure der Nukleotide 1027-1965 aus SEQ ID NO:3. Ebenso geeignet sind Nukleinsäuren, die gegenüber dieser Nukleinsäure vorzugsweise am 5'-Ende beispielsweise um ca. 60 Nukleotide verkürzt oder verlängert sind. Besonders bevorzugt ist eine verlängerte Nukleinsäure der Nukleotide 970-1965, welche einer Proform der Protease entspricht. Verkürzte Nukleinsäuren entsprechen proteolytisch, vorzugsweise autoproteolytisch prozessierten Proteinen.A nucleic acid of nucleotides 1027-1965 from SEQ ID NO: 3 is preferred. Also suitable are nucleic acids which are shortened or extended, for example by approximately 60 nucleotides, at the 5 'end compared to this nucleic acid. An extended nucleic acid of nucleotides 970-1965 which corresponds to a proform of the protease is particularly preferred. Shortened nucleic acids correspond to proteolytically, preferably autoproteolytically processed proteins.
Die Aktivität der neutralen Protease ist dem Fachmann bekannt und bei Mandel et al. (1953) Sparrow und McQuade (1973) und Hefley (1987) beschrieben. Neutrale Protease spaltet sowohl Casein als auch denaturiertes Collagen.The activity of the neutral protease is known to the person skilled in the art and is described in Mandel et al. (1953) Sparrow and McQuade (1973) and Hefley (1987). Neutral protease cleaves both casein and denatured collagen.
Unter Hybridisierung, im Sinne der Erfindung, ist eine Hybridisierung unter dem Fachmann geläufigen, üblichen stringenten Bedingungen zu verstehen, wie sie beispielsweise von J. Sambrook in Molecular Cloning, Cold Spring Harbor Laboratory (1989) und B.D. Harnes, S.G. Higgins, Nucleic Acid Hybridization - A practical approach (1985), IRL-Press, Oxford, England, angegeben sind. Üblicherweise werden zur Hybridisierung die Standardprotokolle verwendet, die in diesen Publikationen beschrieben sind.Hybridization, in the sense of the invention, is to be understood as a hybridization under the usual stringent conditions familiar to the person skilled in the art, as described, for example, by J. Sambrook in Molecular Cloning, Cold Spring Harbor Laboratory (1989) and B.D. Harnes, S.G. Higgins, Nucleic Acid Hybridization - A practical approach (1985), IRL-Press, Oxford, England. The standard protocols described in these publications are usually used for hybridization.
Vorzugsweise wird unter "stringenten Bedingungen" eine Hybridisierung in 6.0 x SSC bei etwa 45°C mit nachfolgendem Waschschritt bei 2,0 x SSC bei 50°C verstanden. Zur Einstel¬ lung der Stringenz kann die Salzkonzentration im Waschschritt beispielsweise von 2,0 x SSC bei 50°C für geringe Stringenz, bis 0,2 x SSC bei 50°C für hohe Stringenz gewählt werden. Zusätzlich kann die Temperatur des Waschschritts zwischen Raumtemperatur (22°C, geringe Stringenz) bis etwa 65°C (hohe Stringenz) eingestellt werden. Die stringenten Hybridisie- rungsbedingungen werden vorzugsweise so gewählt, daß wenigstens eine Homologie von 75 %, vorzugsweise von 90 %, in der Aminosäuresequenz erhalten wird."Stringent conditions" is preferably understood to mean a hybridization in 6.0 x SSC at about 45 ° C. with a subsequent washing step at 2.0 x SSC at 50 ° C. To set the stringency, the salt concentration in the washing step can be selected, for example, from 2.0 x SSC at 50 ° C. for low stringency to 0.2 x SSC at 50 ° C. for high stringency. In addition, the temperature of the washing step can be set between room temperature (22 ° C, low stringency) to about 65 ° C (high stringency). The stringent hybridization Rung conditions are preferably chosen so that at least a homology of 75%, preferably 90%, is obtained in the amino acid sequence.
.Als Nukleinsäure im Sinne der Erfindung ist eine DNA oder RNA geeignet. Dabei ist die RNA komplementär zu einer erfindungsgemäßen DNA. Die erfindungsgemäße Nukleinsäure kann synthetischen, semisynthetischen oder rekombinanten Ursprungs sein.A DNA or RNA is suitable as nucleic acid in the sense of the invention. The RNA is complementary to a DNA according to the invention. The nucleic acid according to the invention can be of synthetic, semisynthetic or recombinant origin.
Es hat sich gezeigt, daß die üblichen Verfahren zur Klonierung für die neutrale Protease nicht anwendbar sind. Wenn nach der Aufreinigung der neutralen Protease Peptidsequenzen ermit¬ telt und hieraus in üblicher Weise degenerierte DNA-Sequenzen abgeleitet werden, werden Sequenzen erhalten, die als Oligonukleotide in PCR-Reaktionen zur Isolierung von Fragmenten führen, die nicht für Proteine codieren, welche eine neutrale Proteaseaktivität haben.It has been shown that the conventional cloning methods cannot be used for the neutral protease. If peptide sequences are determined after the neutral protease has been purified and degenerate DNA sequences are derived therefrom in the usual manner, sequences are obtained which, as oligonucleotides in PCR reactions, lead to the isolation of fragments which do not code for proteins which have a neutral protease activity to have.
Aus der Teilsequenzierung der neutralen Protease wurden beispielsweise die Peptidsequenzen NP23 (SEQ ID NO:8, wobei in Position 3 statt Ala auch Asp stehen kann) und NP44 (SEQ ID NO:4) abgeleitet. Mit degenerierten Primern, die von diesen Peptiden abgeleitet sind, erhält man in der PCR-Reaktion ein 300 bp großes Fragment, das nicht für eine neutrale Protease codiert. Mit einem degenerierten Primer, abgeleitet von Peptid NP44 allein, erhält man ein etwa 400 bp großes Fragment, welches ebenfalls nicht für die neutrale Protease codiert. Auch die Verwendung des Primers 86- 1F (SEQ ID NO.11) basierend auf dem Peptid NP86 (SEQ ID NO: 10) ergibt ein 350 bp-Fragment ohne Bezug zur neutralen Protease.From the partial sequencing of the neutral protease, for example, the peptide sequences NP23 (SEQ ID NO: 8, where Asp can also be in position 3 instead of Ala) and NP44 (SEQ ID NO: 4) were derived. With degenerate primers derived from these peptides, a 300 bp fragment is obtained in the PCR reaction which does not code for a neutral protease. With a degenerate primer, derived from peptide NP44 alone, an approximately 400 bp fragment is obtained which likewise does not code for the neutral protease. The use of the primer 86-1F (SEQ ID NO.11) based on the peptide NP86 (SEQ ID NO: 10) also results in a 350 bp fragment unrelated to the neutral protease.
Daraus ergibt sich, daß das Screenen mit von Peptidsequenzen abgeleiteten Primern im Falle der neutralen Protease nicht ohne weiteres zu brauchbaren Ergebnissen führt.It follows from this that screening with primers derived from peptide sequences does not readily lead to useful results in the case of the neutral protease.
Überraschenderweise ergab sich jedoch bei Kombination der Primer 23F (SEQ ID NO:9)/ 86- 1R (SEQ-ID-NO: 12) ein ca. 320 bp großes Fragment, das zum Gen der neutralen Protease gehört und zum Markieren und Screenen verwendet werden kann. Auch mit Hilfe dieses markierten Stückes war es jedoch noch nicht möglich, Klone mit weiteren Teilen des neutralen Proteasegens zu fischen. Erst durch weitere Modifikation des Klonierungsverfahrens konnte die codierende DNA gefunden werden.Surprisingly, however, when the primers 23F (SEQ ID NO: 9) / 86-1R (SEQ-ID-NO: 12) were combined, a fragment of approximately 320 bp was found which belongs to the neutral protease gene and is used for labeling and screening can be. Even with the help of this marked piece, it was not yet possible to fish clones with further parts of the neutral protease gene. The coding DNA could only be found by further modification of the cloning method.
Die Herstellung der rekombinanten Protease kann nach den dem Fachmann geläufigen Metho¬ den erfolgen. Dazu wird zunächst eine DNA hergestellt, welche in der Lage ist, ein Protein zu produzieren, welches die Aktivität der Protease besitzt. Eine solche DNA, die ausgewählt ist aus der Gruppe umfassendThe recombinant protease can be prepared by the methods familiar to the person skilled in the art. For this purpose, a DNA is first produced, which is able to convert a protein produce, which has the activity of the protease. Such a DNA, which is selected from the group comprising
a) eine DNA der Nukleotide 1027-1965 aus SEQ ID NO: 3 oder einer hierzu komplementären DNA, b) Nukleinsäuren, die mit einer DNA der Nukleotide 1027-1965 aus SEQ ID NO:3 hybridisieren, c) Nukleinsäuren, die ohne die Degeneration des genetischen Codes, mit einer der in a) oder b) genannten Nukleinsäuren hybridisieren würden,a) a DNA of nucleotides 1027-1965 from SEQ ID NO: 3 or a complementary DNA, b) nucleic acids that hybridize with a DNA of nucleotides 1027-1965 from SEQ ID NO: 3, c) nucleic acids that do not degenerate the genetic code, would hybridize with one of the nucleic acids mentioned in a) or b),
wird ausgewählt und in einen Expressionsvektor insertiert. Ein derartiger Vektor enthält zusätzlich zur NP-Sequenz Promotor/Operator-Elemente, die zur Expression der DNA erforderlich sind. Dieser Vektor, der die NP-Sequenz und die Promotor/Operator-Elemente enthält, wird in einen Wirtsstamm transferiert, der in der Lage ist, die DNA von NP zu exprimieren. Die Wirtszelle wird unter Bedingungen, die zur Amplifikation des Vektors geeignet sind, kultiviert und NP gewonnen. Dabei wird durch geeignete Maßnahmen sicherge¬ stellt, daß das Protein eine aktive tertiäre Struktur einnehmen kann, in der es NP-Eigenschaf- ten zeigt.is selected and inserted into an expression vector. In addition to the NP sequence, such a vector contains promoter / operator elements which are required for the expression of the DNA. This vector, which contains the NP sequence and the promoter / operator elements, is transferred to a host strain that is capable of expressing the DNA of NP. The host cell is cultured under conditions suitable for amplifying the vector and NP is obtained. Suitable measures are taken to ensure that the protein can have an active tertiary structure in which it shows NP properties.
Dabei ist es nicht erforderlich, daß das exprimierte Protein die exakte NP-Aminosäuresequenz wie SEQ ID NO: 5 enthält. Ebenso geeignet sind Proteine, welche im wesentlichen die gleiche Sequenz enthalten und analoge Eigenschaften zeigen. SEQ DD NO: l und SEQ ID NO:2 zeigen bevorzugte DNA-Fragmente. Bevorzugt ist eine DNA der Nukleotide 1027-1965 aus SEQ ID NO:3. Ebenso geeignet sind Nukleinsäuren, die gegenüber dieser Sequenz vorzugsweise am 5'-Ende beispielsweise um ca. 60 Nukleotide verkürzt oder verlängert sind. Besonders bevorzugt ist eine verlängerte Nukleinsäure der Nukleotide 970-1965, welche einer Proform der Protease entspricht. Verkürzte Nukleinsäuren entsprechen proteolytisch, vor¬ zugsweise autoproteolytisch prozessierten Proteinen.It is not necessary for the expressed protein to contain the exact NP amino acid sequence as SEQ ID NO: 5. Proteins which contain essentially the same sequence and show similar properties are also suitable. SEQ DD NO: 1 and SEQ ID NO: 2 show preferred DNA fragments. A DNA of nucleotides 1027-1965 from SEQ ID NO: 3 is preferred. Also suitable are nucleic acids that are shortened or extended, for example, by about 60 nucleotides at the 5 'end compared to this sequence. An extended nucleic acid of nucleotides 970-1965 which corresponds to a proform of the protease is particularly preferred. Shortened nucleic acids correspond to proteolytically, preferably autoproteolytically processed proteins.
Auch die Nukleinsäuresequenz des Proteins kann modifiziert sein. Derartige Modifikationen sind beispielsweise:The nucleic acid sequence of the protein can also be modified. Such modifications are, for example:
- Veränderung der Nukleinsäure, um verschiedene Erkennungssequenzen von Restriktionsenzymen zur Erleichterung der Schritte der Ligation, Klonierung und Mutagenese einzuführen - Veränderung der Nukleinsäure zum Einbau von bevorzugten Codons für die Wirtszelle- Change of nucleic acid to introduce different restriction enzyme recognition sequences to facilitate the steps of ligation, cloning and mutagenesis - Change the nucleic acid to incorporate preferred codons for the host cell
- Ergänzung der Nukleinsäure um zusätzliche Operator-Elemente, um die Expression in der Wirtszelle zu optimieren.- Supplementing the nucleic acid with additional operator elements in order to optimize expression in the host cell.
Ein weiterer Gegenstand der Erfindung ist ein Verfahren zur Herstellung eines Polypeptids, welches die Eigenschaften einer NP aus Clostridium histolyticum hat, durch Expression einer exogenen Nukleinsäure in prokaryontischen oder eukaryontischen Wirtszellen und Isolierung des gewünschten Polypeptids, wobei die DNA, welche für das genannte Peptid codiert, ausgewählt ist aus der Gruppe umfassendAnother object of the invention is a method for producing a polypeptide, which has the properties of an NP from Clostridium histolyticum, by expressing an exogenous nucleic acid in prokaryotic or eukaryotic host cells and isolating the desired polypeptide, the DNA coding for said peptide is selected from the group comprising
a) eine DNA der Nukleotide 1027-1965 aus SEQ ID NO: 3 oder eine hierzu komplementäre DNA, b) Nukleinsäuren, die mit einer DNA der Nukleotide 1027-1965 aus SEQ ID NO:3 hybridisieren, c) Nukleinsäuren, die ohne die Degeneration des genetischen Codes, mit einer der in a) oder b) genannten Nukleinsäuren hybridisieren würden.a) a DNA of nucleotides 1027-1965 from SEQ ID NO: 3 or a complementary DNA, b) nucleic acids that hybridize with a DNA of nucleotides 1027-1965 from SEQ ID NO: 3, c) nucleic acids that do not degenerate of the genetic code, would hybridize with one of the nucleic acids mentioned in a) or b).
Vorzugsweise wird eine DNA der Nukleotide 1027-1965 aus SEQ ID NO:3 verwendet. Zur Expression wird ein biologisch funktionelles Plasmid oder ein viraler DNA- Vektor verwendet, der eine erfindungsgemäße Nukleinsäure enthält. Mit diesem Vektor wird eine eukaryontische oder prokaryontische Wirtszelle stabil transformiert oder transfiziert.A DNA of nucleotides 1027-1965 from SEQ ID NO: 3 is preferably used. A biologically functional plasmid or a viral DNA vector which contains a nucleic acid according to the invention is used for expression. With this vector, a eukaryotic or prokaryotic host cell is stably transformed or transfected.
Die Expression des Proteins erfolgt vorzugsweise in Mikroorganismen, insbesondere in Pro- karyonten, und dort in E. coli.The protein is preferably expressed in microorganisms, in particular in prokaryotes, and there in E. coli.
Die Expressionsvektoren müssen einen Promotor enthalten, der die Expression des Proteins im Wirtsorganismus erlaubt. Derartige Promotoren sind dem Fachmann bekannt und sind beispielsweise lac Promotor (Chang et al., Nature 198 (1977) 1056), trp (Goeddel et al., Nuc. Acids Res. 8 (1980) 4057), λPL Promotor (Shimatake et al., Nature 292 (1981) 128) und T5 Promotor (US-Patent Nr. 4,689,406). Ebenfalls geeignet sind synthetische Promotoren wie beispielsweise tac Promotor (US-Patent Nr. 4,551,433). Ebenso geeignet sind gekoppelte Promotorsysteme wie beispielsweise T7-RNA-Polymerase Promotorsystem (Studier et al., J. Mol. Biol. 189 (1986) 113). Ebenso geeignet sind hybride Promotoren aus einem Bacterio- phagen-Promotor und der Operator-Region des Mikroorganismus (EP-A 0267 851). Zusätz¬ lich zum Promotor ist eine effektive Ribosomenbindungsstelle erforderlich. Für E. coli wird diese Ribosomenbindungsstelle als Shine-Dalgamo (SD)-Sequenz bezeichnet (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).The expression vectors must contain a promoter which allows expression of the protein in the host organism. Such promoters are known to the person skilled in the art and are, for example, lac promoter (Chang et al., Nature 198 (1977) 1056), trp (Goeddel et al., Nuc. Acids Res. 8 (1980) 4057), λ PL promoter (Shimatake et al., Nature 292 (1981) 128) and T5 promoter (U.S. Patent No. 4,689,406). Synthetic promoters such as the tac promoter (US Pat. No. 4,551,433) are also suitable. Coupled promoter systems such as, for example, T7 RNA polymerase promoter system (Studier et al., J. Mol. Biol. 189 (1986) 113) are also suitable. Hybrid promoters comprising a bacteriophage promoter and the operator region of the microorganism (EP-A 0267 851) are also suitable. An effective ribosome binding site is required in addition to the promoter. For E. coli referred to this ribosome binding site as the Shine-Dalgamo (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, USA).
Zur Verbesserung der Expression ist es möglich, das Protein als Fusionsprotein zu exprimie- ren. In diesem Fall wird üblicherweise eine DNA, welche für den N-terminalen Teil eines endogenen bakteriellen Proteins oder ein anderes stabiles Protein codiert, an das 5'-Ende der für die NP codierenden DNA fusioniert. Beispiele hierfür sind beispielsweise lacZ (Phillips and Silhavy, Nature 344 (1990) 882 - 884), trpE (Yansura , Meth. Enzymol. 185 (1990) 161 - 166).To improve the expression, it is possible to express the protein as a fusion protein. In this case, a DNA which codes for the N-terminal part of an endogenous bacterial protein or another stable protein is usually attached to the 5 'end of the fused for the NP coding DNA. Examples include lacZ (Phillips and Silhavy, Nature 344 (1990) 882-884), trpE (Yansura, Meth. Enzymol. 185 (1990) 161-166).
Die nach Expression erhaltenen Fusionsproteine werden vorzugsweise mit Enzymen (z.B. Faktor Xa) gespalten (Nagai et al., Nature 309 (1984) 810). Weitere Beispiele für die Spalt¬ stellen sind die IgA-Protease-Spaltstelle (WO 91/11520, EP-A 0 495 398) und die Ubiquitin- Spaltstelle (Miller et al., Bio/Technology 7 (1989) 698).The fusion proteins obtained after expression are preferably cleaved with enzymes (e.g. factor Xa) (Nagai et al., Nature 309 (1984) 810). Further examples of the cleavage sites are the IgA protease cleavage site (WO 91/11520, EP-A 0 495 398) and the ubiquitin cleavage site (Miller et al., Bio / Technology 7 (1989) 698).
Die auf diese Weise in Bakterien exprimierten Proteine werden durch Aufschluß der Bakterien und Proteinisolierung in üblicher Weise gewonnen.The proteins expressed in this way in bacteria are obtained in a conventional manner by digesting the bacteria and isolating the proteins.
In einer weiteren Ausfuhrungsform ist es möglich, die Proteine als aktive Proteine aus den Mikroorganismen zu sekretieren. Hierzu wird vorzugsweise ein Fusionsprodukt verwendet, welches aus der Signalsequenz, die für die Sekretion von Proteinen in den verwendeten Wirts¬ organismen geeignet ist, und der Nukleinsäure, welche für das Protein codiert, besteht. Das Protein wird dabei entweder in das Medium (bei grampositiven Bakterien) oder in den peri- plasmatischen Raum (bei gramnegativen Bakterien) sekretiert. Zwischen der Signalsequenz und der für die NP codierenden Sequenz ist zweckmäßig eine Spaltstelle angebracht, die ent¬ weder bei der Prozessierung oder in einem zusätzlichen Schritt die Abspaltung des Proteins erlaubt. Derartige Signalsequenzen sind beispielsweise ompA (Ghrayeb et al., EMBO J. 3 (1984) 2437), phoA (Oka et al., Proc. Natl. Acad. Sei. USA 82 (1985) 7212).In a further embodiment it is possible to secrete the proteins as active proteins from the microorganisms. For this purpose, a fusion product is preferably used, which consists of the signal sequence which is suitable for the secretion of proteins in the host organisms used and the nucleic acid which codes for the protein. The protein is either secreted into the medium (for gram-positive bacteria) or into the periplasmic space (for gram-negative bacteria). Between the signal sequence and the sequence coding for the NP, a cleavage site is expediently provided, which allows the protein to be split off either during processing or in an additional step. Such signal sequences are, for example, ompA (Ghrayeb et al., EMBO J. 3 (1984) 2437), phoA (Oka et al., Proc. Natl. Acad. Sci. USA 82 (1985) 7212).
Zusätzlich enthalten die Vektoren noch Terminatoren. Terminatoren sind DNA-Sequenzen, die das Ende eines Transkriptionsvorganges signalisieren. Sie zeichnen sich meist durch zwei strukturelle Eigenarten aus: eine umgekehrt repetitive G/C-reiche Region, die intramolekular eine Doppelhelix bilden kann, sowie eine Anzahl von U(bzw. T)-Resten. Beispiele sind trp- Attenuator und Terminator in der DNA des Phagen fd sowie rrnB (Brosius et al., J. Mol. Biol. 148 (1981) 107 - 127). Zusätzlich enthalten die Expressionsvektoren üblicherweise einen selektierbaren Marker, um transformierte Zellen zu selektieren. Derartige selektierbare Marker sind beispielsweise die Resistenzgene für Ampicillin, Chloramphenicol, Erythromycin, Kanamycin, Neomycin und Tetracyclin (Davies et al., Ann. Rev. Microbiol. 32 (1978) 469). Ebenso geeignete selektier¬ bare Marker sind die Gene für essentielle Substanzen der Biosynthese von für die Zelle not¬ wendigen Stoffen wie z.B. Histidin, Tryptophan und Leucin.The vectors also contain terminators. Terminators are DNA sequences that signal the end of a transcription process. They are usually characterized by two structural peculiarities: an inverted repetitive G / C-rich region that can intramolecularly form a double helix, and a number of U (or T) residues. Examples are trp attenuator and terminator in the DNA of phage fd and rrnB (Brosius et al., J. Mol. Biol. 148 (1981) 107-127). In addition, the expression vectors usually contain a selectable marker to select transformed cells. Such selectable markers are, for example, the resistance genes for ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline (Davies et al., Ann. Rev. Microbiol. 32 (1978) 469). Likewise suitable selectable markers are the genes for essential substances of the biosynthesis of substances necessary for the cell, such as histidine, tryptophan and leucine.
Es sind eine Vielzahl von geeigneten bakteriellen Vektoren bekannt. Beispielsweise sind für die folgenden Bakterien Vektoren beschrieben: Bacillus subtilis (Palva et al., Proc. Natl. Acad. Sei. 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 und Streptomyces lividans (US-Patent Nr. 4,747,056).A variety of suitable bacterial vectors are known. For example, vectors are described for the following bacteria: Bacillus subtilis (Palva et al., Proc. Natl. 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 ).
Weitere gentechnologische Verfahren zur Herstellung und Expression von geeigneten Vekto¬ ren sind in J. Sambrook et al., Molecular Cloning: a laboratory manual (1989), Cold Spring Harbor Laboratory Press, New York, N. Y. beschrieben.Further genetic engineering processes for the production 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.
Eine Expression von rekombinanter NP ist außer in prokaryontischen Mikroorganismen auch in Eukaryonten (wie beispielsweise CHO-Zellen, Hefe oder Insektenzellen) möglich. Als eukaryontisches Expressionssystem wird das Hefesystem oder Insektenzellen bevorzugt. Die Expression in Hefe kann über drei Arten von Hefevektoren (integrierende YIp (yeast integrating plasmids)- Vektoren, replizierende YRp (yeast replicon plasmids)- Vektoren und episomale YEp (yeast episomal plasmids)- Vektoren erfolgen. Näheres hierzu ist beispielsweise in S.M. Kingsman et al, Tibtech 5 (1987) 53 - 57 beschrieben.In addition to prokaryotic microorganisms, recombinant NPs can also be expressed in eukaryotes (such as CHO cells, yeast or insect cells). The yeast system or insect cells is preferred as the eukaryotic expression system. Expression in yeast can be via three types of yeast vectors (integrating YIp (yeast integrating plasmids) vectors, replicating YRp (yeast replicon plasmids) vectors and episomal YEp (yeast episomal plasmids) vectors. For more information, for example, in SM Kingsman et al, Tibtech 5 (1987) 53-57.
Ein weiterer Gegenstand der Erfindung ist ein Verfahren zur Auflösung von Zellgewebe und Freisetzung von darin enthaltenen Zellen oder Zellverbänden durch Inkubation des Zellgewe¬ bes mit einer neutralen Protease aus Clostridium histolyticum, welche codiert wird vonAnother object of the invention is a method for dissolving cell tissue and releasing cells or cell assemblies contained therein by incubating the cell tissue with a neutral protease from Clostridium histolyticum, which is encoded by
a) einer DNA der Nukleotide 1027-1965 aus SEQ ID NO: 3 oder einer hierzu komplementären DNA, b) Nukleinsäuren, die mit einer DNA der Nukleotide 1027-1965 aus SEQ ID NO:3 hybridisieren, c) Nukleinsäuren, die ohne die Degeneration des genetischen Codes, mit einer der in a) oder b) genannten Nukleinsäuren hybridisieren würden, und das Produkt einer prokaryontischen oder eukaryontischen Expression einer exogenen DNA ist, bis zur Freisetzung der Zellen oder Zellverbände im gewünschten Umfang und Abtrennung der Zellen oder der Zellverbände von den Zellgewebeanteilen. Diese Protease ist durch die rekombinante Herstellung in einer Wirtszelle, die von Clostridium histolyticum ver¬ schieden ist, frei von anderen Proteinen aus Clostridium histolyticum. Die Abtrennung der Zellen oder Zellverbände von den Zellgewebsanteilen erfolgt bevorzugt durch Zentrifugation mit einem Dichtegradienten.a) a DNA of nucleotides 1027-1965 from SEQ ID NO: 3 or a complementary DNA, b) nucleic acids that hybridize with a DNA of nucleotides 1027-1965 from SEQ ID NO: 3, c) nucleic acids that do not degenerate the genetic code, would hybridize with one of the nucleic acids mentioned in a) or b), and the product is a prokaryotic or eukaryotic expression of an exogenous DNA until the cells or cell assemblies are released to the desired extent and the cells or cell assemblies are separated from the cell tissue portions. This protease is free from other proteins from Clostridium histolyticum due to the recombinant production in a host cell which is different from Clostridium histolyticum. The cells or cell assemblies are separated from the cell tissue portions preferably by centrifugation with a density gradient.
Vorzugsweise wird eine Protease verwendet, die von den Nukleotiden 1027-1965 von SEQ ID NO:3 codiert wird, oder eine um ca. 20 Aminosäuren N-terminal verlängerte oder ver¬ kürzte Form. Besonders bevorzugt ist eine verlängerte Protease, die von einer DNA der Nukleotide 970-1965 aus SEQ ED NO: 3 codiert wird und einer Proform der Protease entspricht. Eine solche verlängerte Protease wird beim Transport durch die Membran ins Periplasma oder ins Medium durch Signalpeptidasen zu einer verkürzten Form (z.B. Protein mit der Aminosäuresequenz SEQ ID NO:5) abgebaut. Ebenso bevorzugt sind N- oder C-terminal verkürzte Formen, welche beispielsweise autokatalytisch entstehen können.A protease is preferably used which is encoded by nucleotides 1027-1965 of SEQ ID NO: 3, or a form which is extended or shortened N-terminally by about 20 amino acids. An extended protease which is encoded by a DNA of nucleotides 970-1965 from SEQ ED NO: 3 and which corresponds to a proform of the protease is particularly preferred. Such an extended protease is degraded to a shortened form (e.g. protein with the amino acid sequence SEQ ID NO: 5) during transport through the membrane into the periplasm or into the medium by signal peptidases. Likewise preferred are shortened N- or C-terminal forms, which can arise, for example, autocatalytically.
Eine Isolierung von Zellen bzw. Zellverbänden aus Geweben (z.B. Pankreas, Leber, Haut, Endothel, Nabelschnur, Knochen) erfolgt im allgemeinen durch Inkubation von Organen, Organteilen oder Geweben mit Enzymen, die die umgebende extrazelluläre Bindegewebe- Matrix auflösen (Inseln: Sutton et al., Transplantation 42 (1986) 689 - 691; Leber: Quibel et al., Anal. Biochem. 154 (1986) 26 - 28; Knochen: Hefley et al., J. Bone Mineral Res. 2 (1987) 505 - 516.Isolation of cells or cell groups from tissues (e.g. pancreas, liver, skin, endothelium, umbilical cord, bone) is generally carried out by incubating organs, organ parts or tissues with enzymes that dissolve the surrounding extracellular connective tissue matrix (Islands: Sutton et al., Transplantation 42 (1986) 689-691; liver: Quibel et al., Anal. Biochem. 154 (1986) 26-28; bone: Hefley et al., J. Bone Mineral Res. 2 (1987) 505 - 516.
Die erfindungsgemäße Proteinase kann auch bei der Präparation von Muskelzellen (Maruyama et al„ J. Pharmacol. Methods 19, 1988, 155 - 164), Fettzellen (Vendrell & Alemany, J. Biochem. Biophys. Methods 16, 1988, 49 - 54), Ovar- und Uterusgewebe (Marcus et al., Endocrine Res. 10, 1984, 151 - 162), Epithelzellen (Kaunitz, Am. J. Physiol. 254, 1988, 6502 - 6512), Herzzellen (Haworth et al., Cell Calcium 10, 1989, 57 - 62) und Plazenta¬ gewebe (Morrish & Siy, Endocrine Res. 12, 1986, 229 - 253) verwendet werden.The proteinase according to the invention can also be used in the preparation of muscle cells (Maruyama et al “J. Pharmacol. Methods 19, 1988, 155-164), fat cells (Vendrell & Alemany, J. Biochem. Biophys. Methods 16, 1988, 49-54) , Ovarian and uterine tissue (Marcus et al., Endocrine Res. 10, 1984, 151-162), epithelial cells (Kaunitz, Am. J. Physiol. 254, 1988, 6502-6512), heart cells (Haworth et al., Cell Calcium 10, 1989, 57-62) and placenta tissue (Morrish & Siy, Endocrine Res. 12, 1986, 229-253) can be used.
Eine Gewebs-Desintegration kann auch durch Perfusion des gesamten Organs (Ricordi et al, Diabetes 37 (1988) 413 - 420) mit Enzymlösung erfolgen. Entscheidend neben der Zusam¬ mensetzung des Enzymgemisches ist dabei die Dauer, der pH-Wert und die Temperatur des Verdaus sowie auch die mechanische Einwirkung, z.B. durch Schütteln und Zusatz von Metallkugeln. Da extrazelluläre Bindegewebsmatrix oft einen hohen Collagen-Anteil aufweist, kommt den Collagenasen und der neutralen Protease eine besondere Rolle zu. (Wolters, Hormone and Metabolie Research 26 (1994) p. 80)Tissue disintegration can also be achieved by perfusing the entire organ (Ricordi et al, Diabetes 37 (1988) 413-420) with enzyme solution. In addition to the composition of the enzyme mixture, the duration, the pH value and the temperature of the digestion, and also the mechanical action, for example by shaking and adding, are decisive Metal balls. Since extracellular connective tissue matrix often has a high proportion of collagen, the collagenases and the neutral protease play a special role. (Wolters, Hormone and Metabolie Research 26 (1994) p. 80)
Vorzugsweise wird das erfindungsgemäße Verfahren zur Isolierung von Inseln oder Inselzel¬ len aus Pankreasgewebe verwendet.The method according to the invention is preferably used for isolating islets or islet cells from pancreatic tissue.
Weitere bevorzugte Anwendungsmöglichkeiten sind die Gewinnung von Zellen aus Geweben aller Art zur Anlage von Zellkulturen oder zur Gewinnung von Zellen, die zu gen- oder zell¬ therapeutischen Zwecken eingesetzt werden (Cell engineering) Ebenfalls angewendet werden können die erfindungsgemäße Proteinase und das erfindungsgemäße Verfahren zur Dissozia¬ tion von Tumorgewebe, vorzugsweise ex vivo. Dabei werden die so isolierten Tumorzellen nach genetischer Veränderung z.B. in den Patienten zurückgegeben, um eine Immunisierung gegen den Tumor zu bewirken, beispielsweise für eine adoptive Immuntherapie.Further preferred applications are the extraction of cells from tissues of all kinds for the establishment of cell cultures or for the extraction of cells which are used for gene or cell therapeutic purposes (cell engineering). The proteinase according to the invention and the method according to the invention for dissociation can also be used ¬ tion of tumor tissue, preferably ex vivo. After genetic modification, the tumor cells isolated in this way are e.g. returned to the patient to effect immunization against the tumor, for example for adoptive immunotherapy.
Daneben kann der Zusatz weiterer Enzyme, wie Collagenasen, Elastase, Trypsin, Chymotrypsin oder Hyaluronidase für die Qualität des Verdaus von Vorteil sein.In addition, the addition of further enzymes, such as collagenases, elastase, trypsin, chymotrypsin or hyaluronidase, can be advantageous for the quality of the digestion.
Die hinterlegten Plasmide sowie die folgenden Beispiele, Publikationen, das Sequenzprotokoll und die Abbildung erläutern die Erfindung, deren Schutzumfang sich aus den Patentansprüchen ergibt, weiter. Die beschriebenen Verfahren sind als Beispiele zu verstehen, die auch noch nach Modifikation den Gegenstand der Erfindung beschreiben.The deposited plasmids as well as the following examples, publications, the sequence listing and the figure further explain the invention, the scope of which results from the patent claims. The described methods are to be understood as examples which describe the subject matter of the invention even after modification.
Das Plasmid pNP-86-lR/23F wurde am 09.12.94 bei der Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg 1b, D-38124 Braunschweig unter der Nummer DSM 9578 hinterlegt.The plasmid pNP-86-IR / 23F was deposited on December 9, 1994 at the German Collection of Microorganisms and Cell Cultures GmbH (DSM), Mascheroder Weg 1b, D-38124 Braunschweig under the number DSM 9578.
Das Plasmid pUC21-E-NP, das die Basen 933-2100 von SEQ ID NO:3 enthält, wurde am 23.11.95 bei der Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg lb, D-38124 Braunschweig unter der Nummer DSM 10341 hinterlegt.The plasmid pUC21-E-NP, which contains the bases 933-2100 of SEQ ID NO: 3, was released on November 23, 1995 at the German Collection of Microorganisms and Cell Cultures GmbH (DSM), Mascheroder Weg lb, D-38124 Braunschweig under the Number DSM 10341 deposited.
Fig. 1 zeigt eine grobe Restriktionskalte der neutralen Protease.Fig. 1 shows a rough restriction cold of the neutral protease.
In den Sequenzprotokollen bedeuten:In the sequence listing:
SEQ ID NO: 1 DNA-Fragment der neutralen ProteaseSEQ ID NO: 1 DNA fragment of the neutral protease
SEQ ID NO:2 DNA-Fragment der neutralen Protease SEQ ID NO:3 DNA der neutralen Protease mit flankierendenSEQ ID NO: 2 DNA fragment of the neutral protease SEQ ID NO: 3 DNA of the neutral protease with flanking
Regionen SEQ ID NO: 5 Proteinsequenz der neutralen ProteaseRegions SEQ ID NO: 5 Protein sequence of the neutral protease
SEQ ID NO:4, 8, 10, 13, 14 und 16 Peptide zur Ableitung von Primern SEQ ID NO:6, 7, 9, 11, 12, 15, 17-20 Primersequenzen.SEQ ID NO: 4, 8, 10, 13, 14 and 16 peptides for the derivation of primers SEQ ID NO: 6, 7, 9, 11, 12, 15, 17-20 primer sequences.
Beispiel 1example 1
Isolierung von neutraler ProteaseIsolation of neutral protease
Reinigung der NP aus Kulturüberstand von Clostidium histolyticumPurification of NP from culture supernatant from Clostidium histolyticum
Lyophilisat der Collagenase P (BM/Best.-Nr. 1213857) wurde in 5 mM HEPES, pH 7.5, 1 M CaCl2 gelöst und abzentrifugiert. Der Überstand wurde auf eine Q-Sepharose-Säule, die mit demselben Puffer äquilibriert war, aufgepumpt (Beladung: max. 20 mg Lyo/ml Säulenmaterial). Nach Waschen der Säule mit dem Äquilibrierungspuffer bis zum Erreichen der Basislinie wurde die NP mit einem steigenden CaCl2-Gradienten (1 - 150 mM, lOfach) eluiert. Die Fraktionen mit hoher caseinolytischer Aktivität (Resorufin-Casein) wurden vereinigt. Auf dem SDS-Gel ist eine deutliche Bande bei ca. 33 kD zu erkennen, als Verunreinigungen treten neben dem "braunen Pigment" im SDS-Gel sichtbare Banden bei ca. 50 kD (chargenabhängig) sowie im niedermolekularen Bereich (< 10 kD) auf.Lyophilisate of collagenase P (BM / Order No. 1213857) was dissolved in 5 mM HEPES, pH 7.5, 1 M CaCl 2 and centrifuged. The supernatant was pumped onto a Q-Sepharose column, which was equilibrated with the same buffer (loading: max. 20 mg Lyo / ml column material). After washing the column with the equilibration buffer until the baseline was reached, the NP was eluted with an increasing CaCl 2 gradient (1 - 150 mM, 10-fold). The fractions with high caseinolytic activity (resorufin-casein) were pooled. On the SDS gel there is a clear band at approx. 33 kD, visible impurities in addition to the "brown pigment" in the SDS gel are visible bands at approx. 50 kD (depending on the batch) and in the low molecular weight range (<10 kD) .
Die NP-Fraktion wird bei 4°C auf eine Butyl 650 C-Säule (äquilibriert mit 10 mM Tris, pH 7.5, 5 mM CaCl2) aufgezogen. Während sich die Verunreinigungen (braunes Pigment, Doppelbande bei ca. 50 kDa) im Durchbruch befinden, wird die NP unter diesen Bedingungen an das hydrophobe Säulenmaterial gebunden. Eine Elution erfolgte bei 4°C mit 10 mM Tris, pH 8.3, 5 mM CaCl2 und 10 % Isopropanol oder 30 % Isopropanol. Das so erhaltene Protein ist gemäß SDS-PAGE homogen (> 95 % sauber). Diese hochreine NP Präparation (Steigerung der spezifischen Aktivität gegen Resorufin-Casein um den Faktor 100) wurde mit Trypsin verdaut und die Peptide über eine Reversed-Phase-HPLC-Säule (C8) aufgetrennt. Nach Einengen der Peptide bis zur Trockene wurde die Aminosäuresequenz bestimmt.The NP fraction is drawn up at 4 ° C. on a butyl 650 C column (equilibrated with 10 mM Tris, pH 7.5, 5 mM CaCl 2 ). While the impurities (brown pigment, double band at approx. 50 kDa) are breakthrough, the NP is bound to the hydrophobic column material under these conditions. Elution was carried out at 4 ° C. with 10 mM Tris, pH 8.3, 5 mM CaCl 2 and 10% isopropanol or 30% isopropanol. The protein obtained in this way is homogeneous according to SDS-PAGE (> 95% clean). This high-purity NP preparation (increase in the specific activity against resorufin-casein by a factor of 100) was digested with trypsin and the peptides separated on a reversed-phase HPLC column (C8). After the peptides had been concentrated to dryness, the amino acid sequence was determined.
Beispiel 2Example 2
Klonierung von neutraler ProteaseCloning of neutral protease
Von nach der Aufreinigung der neutralen Protease gemäß Beispiel 1 ermittelten Peptidsequen¬ zen ausgehend wird die zugehörige (degenerierte) DNA-Sequenz abgeleitet. Sequenzen, die eine vorteilhafte (geringe) Denaturierung zeigen, werden verwendet, um z.B. via PCR eine markierte DNA Probe zum Screenen von Genbanken herzustellen.The associated (degenerate) DNA sequence is derived from the peptide sequences determined after the purification of the neutral protease according to Example 1. Sequences that show an advantageous (low) denaturation are used, for example, to produce a labeled DNA sample via PCR for screening gene banks.
Besonders geeignet sind z.B. 2 Peptide NP 23 und NP 86, von denen sich folgende Primer ableiten lassen:For example, 2 peptides NP 23 and NP 86, from which the following primers can be derived:
Peptid NP 23 : (SEQ ID NO: 8)Peptide NP 23: (SEQ ID NO: 8)
Primer NP 23F: (SEQ ID NO: 9)Primer NP 23F: (SEQ ID NO: 9)
Primer NP 23R: (SEQ ID NO: 6)Primer NP 23R: (SEQ ID NO: 6)
Peptid NP 86: (SEQ ID NO: 10)Peptide NP 86: (SEQ ID NO: 10)
Primer NP 86-1F: (SEQ ID NO: 11)Primer NP 86-1F: (SEQ ID NO: 11)
Primer NP 86-1R: (SEQ ID NO: 12)Primer NP 86-1R: (SEQ ID NO: 12)
Da am Beginn der Experimente die Lage der beiden Peptide zueinander unbekannt ist, muß man, um ein PCR-Fragment des zugehörigen Gens aus genomischer DNA von Clostridium histolyticum amplifizieren zu können, zwei verschiedene Primerkombinationen verwenden: 23F/86-1R und 23R/86-1F. Eine dieser Kombinationen sollte bei Gelingen des Experiments ein Fragment in der PCR ergeben, die zweite Kombination stellt dann gleichzeitig eine Nega¬ tivkontrolle dar.Since the position of the two peptides relative to one another is unknown at the start of the experiments, two different primer combinations must be used in order to be able to amplify a PCR fragment of the associated gene from Clostridium histolyticum genomic DNA: 23F / 86-1R and 23R / 86- 1F. If the experiment is successful, one of these combinations should result in a fragment in the PCR, the second combination then simultaneously represents a negative control.
Mit Primer NP 23F und NP 86- 1R erhält man tatsächlich nach PCR mit nach herkömmlichen Methoden isolierter DNA aus Clostridium histolyticum ein Fragment von ca. 320 bp Länge. Die Kombination 23R/86-1F ergibt kein Fragment.With primer NP 23F and NP 86-1R, a fragment of approximately 320 bp in length is actually obtained after PCR with DNA isolated from Clostridium histolyticum using conventional methods. The combination 23R / 86-1F does not result in a fragment.
Dieses ca. 320 bp lange Fragment kann gut mit dig-dUTP markiert werden, z.B. ebenfalls in einer PCR Reaktion, und dient als Sonde zur Identifizierung positiver Klone aus einer Gen¬ bank. Die Genbank kann in allgemein bekannter Weise aus Clostridium histolyticum DNA nach Verdau mit Restriktionsenzymen hergestellt werden.This approximately 320 bp long fragment can be labeled well with dig-dUTP, e.g. likewise in a PCR reaction, and serves as a probe for the identification of positive clones from a gene bank. The gene bank can be produced in a generally known manner from Clostridium histolyticum DNA after digestion with restriction enzymes.
Das ca. 320 bp lange Fragment wurde sequenziert und enthält DNA der Sequenz SEQ ID NO:2. Diese Sequenz ist auch im Plasmid pNP-86-lR/23F enthalten, welches bei der Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, 38124 Braunschweig (DSM) unter Nr. 9578 am 09.12.1994 hinterlegt wurde.The approximately 320 bp long fragment was sequenced and contains DNA of the sequence SEQ ID NO: 2. This sequence is also contained in the plasmid pNP-86-IR / 23F, which was deposited with the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg lb, 38124 Braunschweig (DSM) under No. 9578 on December 9, 1994.
Genomische DNA von Clostridium histolyticum wurde mit Hindlll verdaut, und die Fragmente wurden anschließend mit DNA Ligase ligiert. Von dem 320 bp Fragment wurden zwei nach außen gerichtete Primer, NPC5 (SEQ ID NO:5) und NPC6 (SEQ ID NO: 15), abgeleitet und in einer PCR-Reaktion eingesetzt. Es wurde ein 586 bp Fragment (SEQ ID NO:l) erhalten, das den Großteil von SEQ ID NO:2 enthielt. Weitere Stücke des Gens der neutralen Protease konnten auf diesem Wege jedoch nicht gefunden werden.Clostridium histolyticum genomic DNA was digested with HindIII and the fragments were then ligated with DNA ligase. From the 320 bp fragment two outward-directed primers, NPC5 (SEQ ID NO: 5) and NPC6 (SEQ ID NO: 15), were derived and used in a PCR reaction. A 586 bp fragment (SEQ ID NO: 1) was obtained which contained the majority of SEQ ID NO: 2. However, no further pieces of the neutral protease gene could be found in this way.
In dem 586 bp-Fragment sind neben Peptid NP23 und einem Teil von Peptid NP86 auch folgende weitere Peptide aus dem Proteinverdau enthalten, die zur Identifizierung des Leserahmens verwendet werden können:In addition to peptide NP23 and part of peptide NP86, the 586 bp fragment also contains the following further peptides from protein digestion that can be used to identify the reading frame:
NP-NT2: (SEQ ID NO: 13) NP58: (SEQ ID NO: 14)NP-NT2: (SEQ ID NO: 13) NP58: (SEQ ID NO: 14)
Von einem neuen Peptid, NP19 (SEQ ID NO: 16), wurde der PCR-Primer NP-19R (SEQ ID NO: 17) abgeleitet. Eine PCR-Reaktion mit Primer NP-19R und dem aus dem 320 bp Fragment abgeleiteten Primer 416 (SEQ ID NO: 18) ergab ein 488 bp großes Fragment, das weitere Sequenzinformation der neutralen Protease enthielt (ca. 350 bp über SEQ ID 2 hinausgehend). Da der Leserahmen 3' immer noch offen war, mußte jedoch noch ein weiteres kleines Stück des Gens fehlen. Mittels Kartierung des Bereichs des neutrale Protease Gens mit Hilfe von Southern Analysen konnte bereits eine grobe Restriktionskarte erstellt werden (Fig.The PCR primer NP-19R (SEQ ID NO: 17) was derived from a new peptide, NP19 (SEQ ID NO: 16). A PCR reaction with primer NP-19R and the primer 416 (SEQ ID NO: 18) derived from the 320 bp fragment resulted in a 488 bp fragment which contained further sequence information of the neutral protease (approx. 350 bp beyond SEQ ID 2) ). Since the reading frame 3 'was still open, another small piece of the gene was still missing. A rough restriction map could already be created by mapping the area of the neutral protease gene with the help of Southern analyzes (Fig.
1).1).
Zur Isolierung des vollständigen 3 '-Endes des neutrale Protease Gens wurde die genomische DNA von Clostridium histolyticum mit EcoRV und Seal geschnitten, die Bruchstücke wurden im Agarose-Gel aufgetrennt und in einem Southern Blot analysiert. Fragmente im Größenbereich um 1000 bp wurden aus dem Gel isoliert, da auf Grund der Restriktionskarte ein solches DNA-Stück die restliche 3 '-Sequenz enthalten müßte. Die aus dem Gel isolierten DNA-Fragmente wurden ligiert (blunt end). Mit Hilfe der nach außen gerichteten Primer 428 (SEQ ID NO: 19) und 429 (SEQ ID NO:20) wurde wieder eine inverse PCR durchgeführt und ein DNA-Fragment der Größe 950 bp erhalten, das das 3 '-Ende des neutrale Protease Gens sowie weitere 470 bp downstream Sequenz enthielt. Damit war das neutrale Protease Gen vollständig isoliert (SEQ ID NO:3).To isolate the complete 3 'end of the neutral protease gene, the genomic DNA from Clostridium histolyticum was cut with EcoRV and Seal, the fragments were separated in the agarose gel and analyzed in a Southern blot. Fragments in the size range around 1000 bp were isolated from the gel, since such a piece of DNA would have to contain the remaining 3 'sequence based on the restriction map. The DNA fragments isolated from the gel were ligated (blunt end). With the help of the outwardly directed primers 428 (SEQ ID NO: 19) and 429 (SEQ ID NO: 20), an inverse PCR was carried out again and a DNA fragment of size 950 bp was obtained which had the 3 'end of the neutral protease Gens and a further 470 bp downstream sequence contained. The neutral protease gene was thus completely isolated (SEQ ID NO: 3).
Das Plasmid pUC21-E-NP, das die Basen 933 - 2100 von SEQ ID NO:3 enthält, wurde am 23.11.95 bei der Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig, unter der Nr. DSM 10341 hinterlegt. Beispiel 3The plasmid pUC21-E-NP, which contains the bases 933-2100 of SEQ ID NO: 3, was released on November 23, 1995 at the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg lb, D-38124 Braunschweig, under the no. DSM 10341 deposited. Example 3
Expression von neutraler ProteaseExpression of neutral protease
DNA-Fragmente, die das Gen für neutrale Protease oder Teile davon enthalten, werden in geeigneter Weise an den Enden modifiziert (z.B. via PCR) und als Ganzes oder in Kombina¬ tion in einen Expressionsvektor für E. coli eingesetzt. Als Promotoren werden bevorzugt sol¬ che verwendet, die gut reguliert werden können wie z.B. lac, tac, trc, mgl; Verwendung anderer Promotoren ist jedoch durchaus denkbar. Zur Sekretion kann entweder das eigene Signalpeptid der neutralen Protease oder ein heterologes (wie z.B. mgl, PhoA) verwendet werden.DNA fragments which contain the gene for neutral protease or parts thereof are suitably modified at the ends (e.g. via PCR) and used as a whole or in combination in an expression vector for E. coli. Preferred promoters are those which can be regulated well, e.g. lac, tac, trc, mgl; However, using other promoters is quite conceivable. Either the neutral protease's own signal peptide or a heterologous one (such as mgl, PhoA) can be used for secretion.
Ein E. coli Stamm, der mit einem Expressionsplasmid transformiert worden ist, wird entweder in Minimalmedium oder LB Medium unter Antibiotika-Selektion über Nacht bei 30 oder 37°C angezogen (z.B. in 5 ml Kultur). Die Übernachtkultur wird in ein größeres Volumen (z.B. 1 1) überimpft und weiterwachsen gelassen. Verwendet man dieses Volumen bereits zur Biomassengewinnung kann man z.B. bei Verwendung eines lac-Promoters bei einer OD-550 von 0.2 bis 2 mit IPTG induzieren und die Zellen weiterwachsen lassen, bis die OD und/oder die gebildete Enzymaktivität nicht weiter zunimmt. Zu diesem Zeitpunkt wird abzentrifugiert und die Biomasse zur Aufreinigung von neutraler Protease verwendet. Es kann sich aber auch ein erheblicher Teil der neutralen Protease- Aktivität im Medium befin¬ den; dann wird auch das Medium gesammelt und neutrale Protease daraus aufgereinigt.An E. coli strain that has been transformed with an expression plasmid is grown either in minimal medium or LB medium with antibiotic selection overnight at 30 or 37 ° C (e.g. in 5 ml culture). The overnight culture is inoculated into a larger volume (e.g. 1 1) and allowed to continue growing. If this volume is already used for biomass production, e.g. when using a lac promoter at an OD-550 of 0.2 to 2, induce with IPTG and let the cells continue to grow until the OD and / or the enzyme activity formed no longer increases. At this point, centrifugation is carried out and the biomass is used to purify neutral protease. However, a considerable part of the neutral protease activity can also be found in the medium; then the medium is also collected and neutral protease is purified therefrom.
Beispiel 4Example 4
Inselisolierung aus Schweine-PankreasIsland isolation from pig pancreas
Aus einem frisch geschlachteten Schwein wird der Pankreas präpariert und in eiskaltem HBSS-Puffer (Gibco) bis zur weiteren Verarbeitung gekühlt. In den Ductus pancreaticus wird eine Braunüle eingeführt und befestigt, die Dichtigkeit des Pankreas wird mit HBSS-Puffer geprüft. Eine Enzymlösung in HBSS-Puffer + Ca2+, die rekombinante neutrale Protease aus Clostridium histolyticum allein oder in Mischung mit gereinigter Collagenase Typ I oder II enthält, wird eingespritzt. Der so behandelte Pankreas wird an die ebenfalls obige Enzym¬ lösung enthaltende Perfusionseinheit (diskontinuierliche Perfusion) angeschlossen. Der Verdau erfolgt zwischen 4°C und 37°C in einem Zeitraum von 5 bis 120 Minuten, wobei die im Gefäß vorhandene Enzymlösung ständig in den Pankreas hineingepumpt wird. Nach der als optimal angenommenen Zeit (üblicherweise 20 bis 30 Minuten, bis Inseln freigesetzt sind) wird die Pumpe gestoppt und das den Pankreas enthaltende Gefäß 3 bis 20 Minuten lang vorsichtig per Hand geschüttelt. Vorher zugegebene Metallkugeln erleichtern zusätzlich die mechanische Dissoziation des Gewebes und die Freisetzung der Inseln aus dem umgebenden exokrinen Gewebe. Der Verlauf des Verdaus wird mikroskopisch nach Dithizonfärbung von in regelmäßigen Abständen entnommenen Proben kontrolliert.The pancreas is prepared from a freshly slaughtered pig and cooled in ice-cold HBSS buffer (Gibco) until further processing. A Braunulse is inserted and fastened into the pancreatic duct, the tightness of the pancreas is checked with HBSS buffer. An enzyme solution in HBSS buffer + Ca2 + , which contains recombinant neutral protease from Clostridium histolyticum alone or in a mixture with purified collagenase type I or II, is injected. The pancreas treated in this way is connected to the perfusion unit (discontinuous perfusion) which also contains the above enzyme solution. The digestion takes place between 4 ° C and 37 ° C in a period of 5 to 120 minutes, the enzyme solution in the vessel being constantly pumped into the pancreas. After the time assumed to be optimal (usually 20 to 30 minutes until islands are released), the pump is stopped and the vessel containing the pancreas is carefully cauterized for 3 to 20 minutes Handshake. Metal balls added beforehand additionally facilitate the mechanical dissociation of the tissue and the release of the islands from the surrounding exocrine tissue. The course of the digestion is checked microscopically after dithizone staining of samples taken at regular intervals.
Der Verdau wird durch Zugabe von eiskaltem HBSS/10 % FKS (fötales Kälberserum) gestoppt und die Suspension durch ein Sieb (Maschengröße 300 μm) filtriert, um die Grob¬ teile abzutrennen. Die im Durchfluß befindlichen Inseln werden 10 Minuten bei 100 g in 250 ml Nalgene-Rundbodenflaschen abzentrifugiert. Der Überstand wird abgenommen und das die Inseln enthaltende Pellet in 50 ml FKS resuspendiert.The digestion is stopped by adding ice-cold HBSS / 10% FCS (fetal calf serum) and the suspension is filtered through a sieve (mesh size 300 μm) in order to separate the large parts. The islands in the flow are centrifuged at 100 g in 250 ml Nalgene round-bottom bottles for 10 minutes. The supernatant is removed and the pellet containing the islets is resuspended in 50 ml FCS.
Eine weitere Aufreinigung der Inseln kann über einen per Hand hergestellten Dichtegradienten erfolgen. In 250 ml Nalgene-Rundbodenflaschen werden zunächst 7 ml der Inselsuspension gegeben. Diese wird zunächst mit 93 ml einer Ficoll-Lösung® (φ = 1.077 g/cm-*), darauffol¬ gend mit 50 ml Medium (RPMI 1640) überschichtet. Diese Gradienten werden im Ausschwingrotor bei 100 g für 10 Minuten zentrifugiert. Fraktionen έ 10 ml werden abge¬ nommen, die Größe, Reinheit und Ausbeute der mit Dithiozon gefärbten Inseln wird in jeder Fraktion mikroskopisch oder mit Hilfe der Bildanalytik bestimmtA further purification of the islands can be done using a density gradient made by hand. First, 7 ml of the island suspension are placed in 250 ml round-bottom Nalgene bottles. This is first covered with 93 ml of a Ficoll solution® (φ = 1,077 g / cm- *), then with 50 ml of medium (RPMI 1640). These gradients are centrifuged in the swing-out rotor at 100 g for 10 minutes. Fractions έ 10 ml are taken off, the size, purity and yield of the islands stained with dithiozone are determined microscopically in each fraction or with the aid of image analysis
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(1) ALLGEMEINE ANGABEN:(1. GENERAL INFORMATION:
(i) ANMELDER:(i) APPLICANT:
(A) NAME: BOEHRINGER MANNHEIM GMBH(A) NAME: BOEHRINGER MANNHEIM GMBH
(B) STRASSE: Sandhofer Str. 116(B) STREET: Sandhofer Str. 116
(C) ORT: Mannheim(C) LOCATION: Mannheim
(E) LAND: Deutschland(E) COUNTRY: Germany
(F) POSTLEITZAHL: D-68305(F) POSTAL NUMBER: D-68305
(G) TELEFON: 08856/60-3446 (H) TELEFAX: 08856/60-3451(G) TELEPHONE: 08856 / 60-3446 (H) TELEFAX: 08856 / 60-3451
(ii) BEZEICHNUNG DER ERFINDUNG: Rekombinante Proteinase aus Clostridium histolyticum und ihre Verwendung zur Isolierung von Zellen und Zellverbaenden(ii) DESCRIPTION OF THE INVENTION: Recombinant proteinase from Clostridium histolyticum and its use for the isolation of cells and cell assemblies
(iii) ANZAHL DER SEQUENZEN: 20(iii) NUMBER OF SEQUENCES: 20
(iv) COMPUTER-LESBARE FASSUNG:(iv) COMPUTER READABLE VERSION:
(A) DATENTRÄGER: Floppy disk(A) DISK: Floppy disk
(B) COMPUTER: IBM PC compatible(B) COMPUTER: IBM PC compatible
(C) BETRIEBSSYSTEM: PC-DOS/MS-DOS(C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30B (EPA)(D) SOFTWARE: Patentin Release # 1.0, Version # 1.30B (EPA)
(vi) DATEN DER URANMELDUNG:(vi) DATA OF THE URN REGISTRATION:
(A) ANMELDENUMMER: DE P 44 45 891.6(A) REGISTRATION NUMBER: DE P 44 45 891.6
(B) ANMELDETAG: 22-DEC-1994(B) REGISTRATION DAY: 22-DEC-1994
(2) ANGABEN ZU SEQ ID NO: 1:(2) INFORMATION ON SEQ ID NO: 1:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LANGE: 586 Basenpaare(A) LONG: 586 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Doppelstrang(C) STRAND FORM: double strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Genom-DNA(ii) MOLECULE TYPE: Genomic DNA
(ix) MERKMAL:(ix) FEATURE:
(A) NAME/SCHLÜSSEL: CDS(A) NAME / KEY: CDS
(B) LAGE:18..584(B) LOCATION: 18..584
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 1:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
AAGCTTCATT TTGGCATATG TTTGTARGTG CTGAAAATGG AAAGATAGTG GATAAGTATA 60AAGCTTCATT TTGGCATATG TTTGTARGTG CTGAAAATGG AAAGATAGTG GATAAGTATA 60
ATGCTTTATC ATGCCAAGCT ACACATGCTC AAGTAAGAGG AGTTAATAGC AGTGGAGAGC 120ATGCTTTATC ATGCCAAGCT ACACATGCTC AAGTAAGAGG AGTTAATAGC AGTGGAGAGC 120
ATAAAATCCT AAATGGTATG TTTGAAAATG GAAGATATTT TTTAGCAGAT TCCACCAGAC 180 CTTCAAATGG ATATATATTA ACATATGATG CTAATAACCA AGAGTATGGT TTCCCAGGTA 240ATAAAATCCT AAATGGTATG TTTGAAAATG GAAGATATTT TTTAGCAGAT TCCACCAGAC 180 CTTCAAATGG ATATATATTA ACATATGATG CTAATAACCA AGAGTATGGT TTCCCAGGTA 240
GCTTATTTAG TAATTTAACA GGCATTTTTC GTAGTGATAG ACCAAAGGCA GGAGTAGATG 300GCTTATTTAG TAATTTAACA GGCATTTTTC GTAGTGATAG ACCAAAGGCA GGAGTAGATG 300
CTCACCATAA TCTAACTCMA GTATATGATT ATTATAAAAA TGTTTTAAAT AGAGATAGTT 360CTCACCATAA TCTAACTCMA GTATATGATT ATTATAAAAA TGTTTTAAAT AGAGATAGTT 360
TTGATGGAAA AGGTGCTAGT ATAATATCTT CTGTGCATTG TAGGAAATAA TTTAAATAAT 420TTGATGGAAA AGGTGCTAGT ATAATATCTT CTGTGCATTG TAGGAAATAA TTTAAATAAT 420
GCTTTCTGGA ATGGTAGACA AATACTTTTT GGTGATGGAG ACGGAGTTAC ATTTAGTAAC 480GCTTTCTGGA ATGGTAGACA AATACTTTTT GGTGATGGAG ACGGAGTTAC ATTTAGTAAC 480
CTAGCAAAAT GTTTAGAAGT TACTGCCCAT GAATTTACAC ATGCAGTTAC TCAAAGTACT 540CTAGCAAAAT GTTTAGAAGT TACTGCCCAT GAATTTACAC ATGCAGTTAC TCAAAGTACT 540
GCAGGTCTAG AATATAGATT TCAATCTGGT GCTCTAAATG AAGCTT 586GCAGGTCTAG AATATAGATT TCAATCTGGT GCTCTAAATG AAGCTT 586
(2) ANGABEN ZU SEQ ID NO: 2:(2) INFORMATION ON SEQ ID NO: 2:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 329 Basenpaare(A) LENGTH: 329 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Doppelstrang(C) STRAND FORM: double strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Genom-DNA(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 2:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
GGCAGGAGTA GATGCTCACC ATAATCTAAC TCMAGTATAT GATTATTATA AAAATGTTTT 60GGCAGGAGTA GATGCTCACC ATAATCTAAC TCMAGTATAT GATTATTATA AAAATGTTTT 60
AAATAGAGAT AGTTTTGATG GAAAAGGTGC TAGTATAATA TCTTCTGTGC ATTGTAGGAA 120AAATAGAGAT AGTTTTGATG GAAAAGGTGC TAGTATAATA TCTTCTGTGC ATTGTAGGAA 120
ATAATTTAAA TAATGCTTTC TGGAATGGTA GACAAATACT TTTTGGTGAT GGAGACGGAG 180ATAATTTAAA TAATGCTTTC TGGAATGGTA GACAAATACT TTTTGGTGAT GGAGACGGAG 180
TTACATTTAG TAACCTAGCA AAATGTTTAG AAGTTACTGC CCATGAATTT ACACATGCAG 240TTACATTTAG TAACCTAGCA AAATGTTTAG AAGTTACTGC CCATGAATTT ACACATGCAG 240
TTACTCAAAG TACTGCAGGT CTAGAATATA GATTTCAATC TGGTGCTCTA AATGAAGCTT 300TTACTCAAAG TACTGCAGGT CTAGAATATA GATTTCAATC TGGTGCTCTA AATGAAGCTT 300
TTTCTGATAT TTTAGGTATA GCTGTTCAC 329TTTCTGATAT TTTAGGTATA GCTGTTCAC 329
(2) ANGABEN ZU SEQ ID NO: 3:(2) INFORMATION ON SEQ ID NO: 3:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 2428 Basenpaare(A) LENGTH: 2428 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: cDNA (ix) MERKMAL:(ii) MOLECULE TYPE: cDNA (ix) FEATURE:
(A) NAME/SCHLÜSSEL: sig_peptide(A) NAME / KEY: sig_peptide
(B) LAGE:970..1026(B) LOCATION: 970..1026
(ix) MERKMAL:(ix) FEATURE:
(A) NAME/SCHLÜSSEL: mat_ >eptide(A) NAME / KEY: mat_> eptide
(B) LAGE:1027..1965(B) LOCATION: 1027..1965
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 3: GACTCTATTG GAGCACTAAT AGGAATTATA ATTATAACAA TTTTATTTAG AAAGAAAAAT 60 GGTTAGAGAG CTTGCTATGA CTTATGTTAT ATGTCATAGC ATTTTTGTTT TATAAGAGGA 120 TTATTAGGAA ATATTACGGG AATCAAAATA AAATCAATAG AATTTAATGT AAATTTTAAC 180 TTAAAAATAT AAACTGAATA TAAAATATAC AAAAACCGGA AAATAATTAG TGAGAATGTT 240 GAGAAAAATT ACAAAAAGTG TATNTACTTT ACCATTTATT AGTACTACAA TAGGGTTATA 300 AATAATAAMG AGGAGGAGTA AAATGAAAAA AAATTTNNNN NNNNNNNNNN NNNNNNNNNN 360 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 420 1WNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNT GCTCTAAATG 480 AAGCTTTTTC TGTATTAAAA ACAGATTTAG AAAAAACCAA GAATATAAAA TCTAATAATA 540 AGGAGGGGGA TGATGTAACA AAAGTAGTTA AGAGTGCTTT AAAAGAAGAA GCCAATTTAG 600 GAGATTTTAA GGGTTGATAA TAAAGAAACT GATGTAAAAG GTAAAAAGCA CTTGCGTTTC 660 ACAAATGTTA TAGATGGTAT TCCTGTATAT GGTAGTCAAG TTATAATTCA TACTAATAAA 720 GATGGACAAG TATATAGCGT AAATGGAAAA GTAGATAAAC AGCCTAAAGC TCAATCTTTT 780 AAGAACCGTG TAAGGATTAA GGACGATAAA GCTATTAAAA TAGCAGAAGA CAGTTTAGGT 840 AAGGAAATAA AGAAAAACAA AAATTATCAT TCTGAAAGTA AGTTGTACCT ATACAAGGTT 900 AATGGAGATT TGCAACCTGT GTATTTGGTA AAGATATCAT CTACAGAACC AGAAGC.TTCA 960 TTTTGGCATA TGTTTGTAAG TGCTGAAAAT GGAAAGATAG TYGATAAGTA TAATGCTTTA 1020 TCATGCCAAG CTACACATGC TCAAGTAAGA GGAGTTAATA GCAGTGGAGA GCATAAAATC 1080 TTAAATGGTA TGTTTGAAAA TGGAAGATAT TTTTTAGCAG ATTCAACAAG ACCTTCAAAT 11 0 GGATATATAT TAACATATGA TGCTAATAAC CAAGAGTATG GTTTCCCAGG TAGCTTATTT 1200 AGTAATTTAA CAGGCATTTT TCGTAGTGAT AGACAAAAGG CAGGAGTAGA TGCTCACCAT 1260 AATCTAACTC AAGTATATGA TTATTATAAA AATGTTTTAA ATAGAGATAG TTTTGATGGA 1320 AAAGGTGCTA GTATAATATC TTCTGTGCAT GTAGGAAATA ATTTAAATAA TGCTTTCTGG 1380 AATGGTAGAC AAATACTTTT TGGTGATGGA GACGGAGTTA CATTTAGTAA CCTAGCAAAA 1 40 TGTTTAGAAG TTACTGCCCA TGAATTTACA CATGCAGTTA CTCAAAGTAC TGCAGGTCTA 1500 GAATATAGAT TTCAATCTGG TGCTCTAAAT GAAGCTTTTT CTGATATTTT AGGTATAGCT 1560 GTTCACAGTG ATCCAAATGA TTGGGAAATT GGAGAAGATA TATACACTCC TAATGTAGCA 1620 GGAGATGCTT TAAGAAGTAT GTCAAATCCT AGATTATATA GACAACCAGA CCATATGAAG 1680 GACTATTTAT ATTGGGATTA TTCAATGGAT AAAGGTGGAG TTCATTATAA TTCAGGTATT 1740 CCAAATAAAG CAGCTTATTT GATGGGAAAA GAAGTTGGAA AAGATTCAAT GGCTAAAATT 1800 TATTATCATG CTTTAGTGAA TTATTTAACT CCTCAAAGTA CATTTGAAGA TGCTAGAAAT 1860 GCAGTAGTAT CATCTGCAAT AGATTTACAT GGTGAGAATA GTAAAGAACA TAAACTTGCT 1920 ATAAAATCTT GGGCAGATGT AGGAGTTGGA GAAGAGGCAG TAAGATAATA GAGAATATGA 1980 AGGATTCCAT TATAATAAAT ATATAATGCC TGTTTTTGAT AGATTAAGTA ATACCATAAA 2040 GTAGAGAATA TAAAAAATAA AAATCTACTG CATTGTATTT TAGATAAATA GGTGCGGAAT 2100 ATAGAACAAG CTAAMTTATA TTAAAAATAA GTATAGGAAT ATAATTAATA GGTAAGGTAA 2160 ATCATTTTTC TAAGGTAGTT GCAGTAGGTA GTATAAAGTA TTAGTAGTAG AGTATATTAG 2220 TTAAAGGAAA AAATCCCTCA CATATAAAAA TACGCTATGT ATATTTGTTA CCTAAAAATT 2280 GAATTATAAA AAAAAGGTGT CTGRAGGCTA ADATAAAACC TTTCGGCACC TTTTTACATT 2340 ACCAGTTATT ATAGTGGATY CTTTCTTTAT CCAATCTATC GTAATGTTTT TTTTCYTCAT 2400 TAGGATACTG CAGGTCTAGA ATATAGAT 2 28(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GACTCTATTG GAGCACTAAT AGGAATTATA ATTATAACAA TTTTATTTAG AAAGAAAAAT 60 GGTTAGAGAG CTTGCTATGA CTTATGTTAT ATGTCATAGC ATTTTTGTTT TATAAGAGGA 120 TTATTAGGAA ATATTACGGG AATCAAAATA AAATCAATAG AATTTAATGT AAATTTTAAC 180 TTAAAAATAT AAACTGAATA TAAAATATAC AAAAACCGGA AAATAATTAG TGAGAATGTT 240 GAGAAAAATT ACAAAAAGTG TATNTACTTT ACCATTTATT AGTACTACAA TAGGGTTATA 300 AATAATAAMG AGGAGGAGTA AAATGAAAAA AAATTTNNNN nnnnnnnnnn nnnnnnnnnn 360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 420 1WNNNNNNNN nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn NNNNNNNNNT GCTCTAAATG 480 AAGCTTTTTC TGTATTAAAA ACAGATTTAG AAAAAACCAA GAATATAAAA TCTAATAATA 540 AGGAGGGGGA TGATGTAACA AAAGTAGTTA AGAGTGCTTT AAAAGAAGAA GCCAATTTAG 600 GAGATTTTAA GGGTTGATAA TAAAGAAACT GATGTAAAAG GTAAAAAGCA CTTGCGTTTC 660 ACAAATGTTA TAGATGGTAT TCCTGTATAT GGTAGTCAAG TTATAATTCA TACTAATAAA 720 GATGGACAAG TATATAGCGT AAATGGAAAA GTAGATAAAC AGCCTAAAGC TCAATCTTTT 780 AAGAACCGTG TAAGGATTAA GGACGATAAA GCTATTAAAA TAGCAGA AGA CAGTTTAGGT 840 AAGGAAATAA AGAAAAACAA AAATTATCAT TCTGAAAGTA AGTTGTACCT ATACAAGGTT 900 AATGGAGATT TGCAACCTGT GTATTTGGTA AAGATATCAT CTACAGAACC AGAAGC.TTCA 960 TTTTGGCATA TGTTTGTAAG TGCTGAAAAT GGAAAGATAG TYGATAAGTA TAATGCTTTA 1020 TCATGCCAAG CTACACATGC TCAAGTAAGA GGAGTTAATA GCAGTGGAGA GCATAAAATC 1080 TTAAATGGTA TGTTTGAAAA TGGAAGATAT TTTTTAGCAG ATTCAACAAG ACCTTCAAAT 11 0 GGATATATAT TAACATATGA TGCTAATAAC CAAGAGTATG GTTTCCCAGG TAGCTTATTT 1200 AGTAATTTAA CAGGCATTTT TCGTAGTGAT AGACAAAAGG CAGGAGTAGA TGCTCACCAT 1260 AATCTAACTC AAGTATATGA TTATTATAAA AATGTTTTAA ATAGAGATAG TTTTGATGGA 1320 AAAGGTGCTA GTATAATATC TTCTGTGCAT GTAGGAAATA ATTTAAATAA TGCTTTCTGG 1380 AATGGTAGAC AAATACTTTT TGGTGATGGA GACGGAGTTA CATTTAGTAA CCTAGCAAAA 1 40 TGTTTAGAAG TTACTGCCCA TGAATTTACA CATGCAGTTA CTCAAAGTAC TGCAGGTCTA 1500 GAATATAGAT TTCAATCTGG TGCTCTAAAT GAAGCTTTTT CTGATATTTT AGGTATAGCT 1560 GTTCACAGTG ATCCAAATGA TTGGGAAATT GGAGAAGATA TATACACTCC TAATGTAGCA 1620 GGAGATGCTT TAAGAAGTAT GTCAAATCCT AGATTATATA GACAACCAGA CCATATGAAG 1680 GACTATTTAT ATTGGGATTA TTCAATGGAT AAAGGTGGAG TTCATTATAA TTCAGGTATT 1740 CCAAATAAAG CAGCTTATTT GATGGGAAAA GAAGTTGGAA AAGATTCAAT GGCTAAAATT 1800 TATTATCATG CTTTAGTGAA TTATTTAACT CCTCAAAGTA CATTTGAAGA TGCTAGAAAT 1860 GCAGTAGTAT CATCTGCAAT AGATTTACAT GGTGAGAATA GTAAAGAACA TAAACTTGCT 1920 ATAAAATCTT GGGCAGATGT AGGAGTTGGA GAAGAGGCAG TAAGATAATA GAGAATATGA 1980 AGGATTCCAT TATAATAAAT ATATAATGCC TGTTTTTGAT AGATTAAGTA ATACCATAAA 2040 GTAGAGAATA TAAAAAATAA AAATCTACTG CATTGTATTT TAGATAAATA GGTGCGGAAT 2100 ATAGAACAAG CTAAMTTATA TTAAAAATAA GTATAGGAAT ATAATTAATA GGTAAGGTAA 2160 ATCATT TTTC TAAGGTAGTT GCAGTAGGTA GTATAAAGTA TTAGTAGTAG AGTATATTAG 2220 TTAAAGGAAA AAATCCCTCA CATATAAAAA TACGCTATGT ATATTTGTTA CCTAAAAATT 2280 GAATTATAAA AAAAAGGTGT CTGRAGGCTA ADATAAAACC TTTCGGCACC TTTTTACATT 2340 ACCAGTTATT ATAGTGGATY CTTTCTTTAT CCAATCTATC GTAATGTTTT TTTTCYTCAT 2400 TAGGATACTG CAGGTCTAGA ATATAGAT 2 28
(2) ANGABEN ZU SEQ ID NO: 4:(2) INFORMATION ON SEQ ID NO: 4:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 8 Aminosäuren(A) LENGTH: 8 amino acids
(B) ART: Aminosäure(B) TYPE: amino acid
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Peptid (xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 4(ii) MOLECULE TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4
Tyr Tyr His Ala Leu Val Asn Tyr 1 5Tyr Tyr His Ala Leu Val Asn Tyr 1 5
(2) ANGABEN ZU SEQ ID NO: 5:(2) INFORMATION ON SEQ ID NO: 5:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 313 Aminosäuren(A) LENGTH: 313 amino acids
(B) ART: Aminosäure(B) TYPE: amino acid
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Protein(ii) MOLECULE TYPE: Protein
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 5:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Gin Ala Thr His Ala Gin Val Arg Gly Val Asn Ser Ser Gly Glu His 1 5 10 15Gin Ala Thr His Ala Gin Val Arg Gly Val Asn Ser Ser Gly Glu His 1 5 10 15
Lys Ile Leu Asn Gly Met Phe Glu Asn Gly Arg Tyr Phe Leu Ala Asp 20 25 30Lys Ile Leu Asn Gly Met Phe Glu Asn Gly Arg Tyr Phe Leu Ala Asp 20 25 30
Ser Thr Arg Pro Ser Asn Gly Tyr Ile Leu Thr Tyr Asp Ala Asn Asn 35 40 45Ser Thr Arg Pro Ser Asn Gly Tyr Ile Leu Thr Tyr Asp Ala Asn Asn 35 40 45
Gin Glu Tyr Gly Phe Pro Gly Ser Leu Phe Ser Asn Leu Thr Gly Ile 50 55 60Gin Glu Tyr Gly Phe Pro Gly Ser Leu Phe Ser Asn Leu Thr Gly Ile 50 55 60
Phe Arg Ser Asp Arg Gin Lys Ala Gly Val Asp Ala His His Asn Leu 65 70 75 80Phe Arg Ser Asp Arg Gin Lys Ala Gly Val Asp Ala His His Asn Leu 65 70 75 80
Thr Gin Val Tyr Asp Tyr Tyr Lys Asn Val Leu Asn Arg Asp Ser Phe 85 90 95Thr Gin Val Tyr Asp Tyr Tyr Lys Asn Val Leu Asn Arg Asp Ser Phe 85 90 95
Asp Gly Lys Gly Ala Ser Ile Ile Ser Ser Val His Val Gly Asn Asn 100 105 110Asp Gly Lys Gly Ala Ser Ile Ile Ser Ser Val His Val Gly Asn Asn 100 105 110
Leu Asn Asn Ala Phe Trp Asn Gly Arg Gin Ile Leu Phe Gly Asp Gly 115 120 125Leu Asn Asn Ala Phe Trp Asn Gly Arg Gin Ile Leu Phe Gly Asp Gly 115 120 125
Asp Gly Val Thr Phe Ser Asn Leu Ala Lys Cys Leu Glu Val Thr Ala 130 135 140Asp Gly Val Thr Phe Ser Asn Leu Ala Lys Cys Leu Glu Val Thr Ala 130 135 140
His Glu Phe Thr His Ala Val Thr Gin Ser Thr Ala Gly Leu Glu Tyr 145 150 155 160His Glu Phe Thr His Ala Val Thr Gin Ser Thr Ala Gly Leu Glu Tyr 145 150 155 160
Arg Phe Gin Ser Gly Ala Leu Asn Glu Ala Phe Ser Asp Ile Leu Gly 165 170 175Arg Phe Gin Ser Gly Ala Leu Asn Glu Ala Phe Ser Asp Ile Leu Gly 165 170 175
Ile Ala Val His Ser Asp Pro Asn Asp Trp Glu Ile Gly Glu Asp Ile 180 185 190 Tyr Thr Pro Asn Val Ala Gly Asp Ala Leu Arg Ser Met Ser Asn Pro 195 200 205Ile Ala Val His Ser Asp Pro Asn Asp Trp Glu Ile Gly Glu Asp Ile 180 185 190 Tyr Thr Pro Asn Val Ala Gly Asp Ala Leu Arg Ser Met Ser Asn Pro 195 200 205
Arg Leu Tyr Arg Gin Pro Asp His Met Lys Asp Tyr Leu Tyr Trp Asp 210 215 220Arg Leu Tyr Arg Gin Pro Asp His Met Lys Asp Tyr Leu Tyr Trp Asp 210 215 220
Tyr Ser Met Asp Lys Gly Gly Val His Tyr Asn Ser Gly Ile Pro Asn 225 230 235 240Tyr Ser Met Asp Lys Gly Gly Val His Tyr Asn Ser Gly Ile Pro Asn 225 230 235 240
Lys Ala Ala Tyr Leu Met Gly Lys Glu Val Gly Lys Asp Ser Met Ala 245 250 255Lys Ala Ala Tyr Leu Met Gly Lys Glu Val Gly Lys Asp Ser Met Ala 245 250 255
Lys Ile Tyr Tyr His Ala Leu Val Asn Tyr Leu Thr Pro Gin Ser Thr 260 265 270Lys Ile Tyr Tyr His Ala Leu Val Asn Tyr Leu Thr Pro Gin Ser Thr 260 265 270
Phe Glu Asp Ala Arg Asn Ala Val Val Ser Ser Ala Ile Asp Leu His 275 280 285Phe Glu Asp Ala Arg Asn Ala Val Val Ser Ser Ala Ile Asp Leu His 275 280 285
Gly Glu Asn Ser Lys Glu His Lys Leu Ala Ile Lys Ser Trp Ala Asp 290 295 300Gly Glu Asn Ser Lys Glu His Lys Leu Ala Ile Lys Ser Trp Ala Asp 290 295 300
Val Gly Val Gly Glu Glu Ala Val Arg 305 310Val Gly Val Gly Glu Glu Ala Val Arg 305 310
(2) ANGABEN ZU SEQ ID NO: 6:(2) INFORMATION ON SEQ ID NO: 6:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 20 Basenpaare(A) LENGTH: 20 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "Primer 23R"(A) DESCRIPTION: / desc = "Primer 23R"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 6: TGNGTNARRT TRTGRTGNGC 20(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: TGNGTNARRT TRTGRTGNGC 20
(2) ANGABEN ZU SEQ ID NO: 7:(2) INFORMATION ON SEQ ID NO: 7:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 19 Basenpaare(A) LENGTH: 19 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "Primer NPC5"(A) DESCRIPTION: / desc = "Primer NPC5"
ERSAT (xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 7: GTGATGGAGA CGGAGTTAC 19REPLACEMENT (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GTGATGGAGA CGGAGTTAC 19
(2) ANGABEN ZU SEQ ID NO: 8:(2) INFORMATION ON SEQ ID NO: 8:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 16 Aminosäuren(A) LENGTH: 16 amino acids
(B) ART: Aminosäure(B) TYPE: amino acid
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Peptid(ii) MOLECULE TYPE: Peptide
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 8:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Gly Val Ala Ala His His Asn Leu Thr Gin Val Tyr Asp Tyr Tyr Lys 1 5 10 15Gly Val Ala Ala His His Asn Leu Thr Gin Val Tyr Asp Tyr Tyr Lys 1 5 10 15
(2) ANGABEN ZU SEQ ID NO: 9:(2) INFORMATION ON SEQ ID NO: 9:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 20 Basenpaare(A) LENGTH: 20 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "Primer 23F"(A) DESCRIPTION: / desc = "Primer 23F"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 9: GCNCAYCAYA AYYTNACNCA 20(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GCNCAYCAYA AYYTNACNCA 20
(2) ANGABEN ZU SEQ ID NO: 10:(2) INFORMATION ON SEQ ID NO: 10:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 24 Aminosäuren(A) LENGTH: 24 amino acids
(B) ART: Aminosäure(B) TYPE: amino acid
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Peptid (xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 10:(ii) MOLECULE TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Ile Ala Val His Ser Asp Pro Asn Asp Trp Glu Ile Gly Glu Asp Ile 1 5 10 15Ile Ala Val His Ser Asp Pro Asn Asp Trp Glu Ile Gly Glu Asp Ile 1 5 10 15
Tyr Thr Pro Asn Val Ala Gly Asp 20Tyr Thr Pro Asn Val Ala Gly Asp 20
(2) ANGABEN ZU SEQ ID NO: 11:(2) INFORMATION ON SEQ ID NO: 11:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 20 Basenpaare(A) LENGTH: 20 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "Primer 86-1F"(A) DESCRIPTION: / desc = "Primer 86-1F"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 11: GAYCCNAAYG AYTGGGARAT 20(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GAYCCNAAYG AYTGGGARAT 20
(2) ANGABEN ZU SEQ ID NO: 12:(2) INFORMATION ON SEQ ID NO: 12:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 20 Basenpaare(A) LENGTH: 20 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc -= "Primer 86-1R"(A) DESCRIPTION: / desc - = "Primer 86-1R"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 12: ATYTCCCART CRTTNGGRTC 20(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: ATYTCCCART CRTTNGGRTC 20
(2) ANGABEN ZU SEQ ID NO: 13:(2) INFORMATION ON SEQ ID NO: 13:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 18 Aminosäuren(A) LENGTH: 18 amino acids
(B) ART: Aminosäure(B) TYPE: amino acid
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Peptid (xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 13:(ii) MOLECULE TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Ala Thr His Ala Xaa Val Arg Gly Val Asn Ser Ser Gly Glu His Lys 1 5 10 15Ala Thr His Ala Xaa Val Arg Gly Val Asn Ser Ser Gly Glu His Lys 1 5 10 15
Ile LeuIle Leu
(2) ANGABEN ZU SEQ ID NO: 14:(2) INFORMATION ON SEQ ID NO: 14:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 16 Aminosäuren(A) LENGTH: 16 amino acids
(B) ART: Aminosäure(B) TYPE: amino acid
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Peptid(ii) MOLECULE TYPE: Peptide
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 14:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Phe Thr His Ala Val Thr Gin Ser Thr Ala Gly Leu Glu Tyr Arg Asp 1 5 10 15Phe Thr His Ala Val Thr Gin Ser Thr Ala Gly Leu Glu Tyr Arg Asp 1 5 10 15
[2) ANGABEN ZU SEQ ID NO: 15:[2) INFORMATION ON SEQ ID NO: 15:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 20 Basenpaare(A) LENGTH: 20 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "Primer NPC6"(A) DESCRIPTION: / desc = "Primer NPC6"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 15: TGTCTACCAT TCCAGAAAGC 20(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: TGTCTACCAT TCCAGAAAGC 20
(2) ANGABEN ZU SEQ ID NO: 16:(2) INFORMATION ON SEQ ID NO: 16:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 11 Aminosäuren(A) LENGTH: 11 amino acids
(B) ART: Aminosäure(B) TYPE: amino acid
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Peptid (xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 16:(ii) MOLECULE TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Ser Trp Ala Asp Val Gly Val Gly Glu Glu Ala 1 5 10Ser Trp Ala Asp Val Gly Val Gly Glu Glu Ala 1 5 10
(2) ANGABEN ZU SEQ ID NO: 17:(2) INFORMATION ON SEQ ID NO: 17:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 19 Basenpaare(A) LENGTH: 19 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "Primer 19-R"(A) DESCRIPTION: / desc = "Primer 19-R"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 17: GCYTCYTCNC CNACNCCTA 19(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: GCYTCYTCNC CNACNCCTA 19
(2) ANGABEN ZU SEQ ID NO: 18:(2) INFORMATION ON SEQ ID NO: 18:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 20 Basenpaare(A) LENGTH: 20 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "Primer 416"(A) DESCRIPTION: / desc = "Primer 416"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 18: GTTACTCAAA GTACTGCAGG 20(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GTTACTCAAA GTACTGCAGG 20
(2) ANGABEN ZU SEQ ID NO: 19:(2) INFORMATION ON SEQ ID NO: 19:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 20 Basenpaare(A) LENGTH: 20 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "Primer 428" (xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 19: CTTGCTATAA AATCTTGGGC 20(A) DESCRIPTION: / desc = "Primer 428" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: CTTGCTATAA AATCTTGGGC 20
(2) ANGABEN ZU SEQ ID NO: 20:(2) INFORMATION ON SEQ ID NO: 20:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 21 Basenpaare(A) LENGTH: 21 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "Primer 429"(A) DESCRIPTION: / desc = "Primer 429"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 20: AAAGCTTCAT TTAGAGCACC C 21 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: AAAGCTTCAT TTAGAGCACC C 21

Claims

Patentansprüche claims
1. Nukleinsäure, welche für ein Protein mit der Aktivität der neutralen Protease aus Clostridium histolyticum codiert, dadurch gekennzeichnet, daß sie ausgewählt ist aus der Gruppe umfassend1. Nucleic acid which codes for a protein with the activity of the neutral protease from Clostridium histolyticum, characterized in that it is selected from the group comprising
a) eine DNA der Nukleotide 1027-1965 aus SEQ ID NO: 3 oder eine hierzu komplementäre DNA,a) a DNA of nucleotides 1027-1965 from SEQ ID NO: 3 or a complementary DNA,
b) Nukleinsäuren, die mit einer DNA der Nukleotide 1027-1965 aus SEQ ID NO:3 hybridisieren,b) nucleic acids which hybridize with a DNA of nucleotides 1027-1965 from SEQ ID NO: 3,
c) Nukleinsäuren, die ohne die Degeneration des genetischen Codes, mit einer der in a) oder b) genannten Nukleinsäuren hybridisieren würden.c) nucleic acids that would hybridize with one of the nucleic acids mentioned in a) or b) without the degeneration of the genetic code.
2. Nukleinsäure nach Anspruch 1 mit einer DNA der Nukleotide 1027-1965 aus SEQ ED NO:3.2. Nucleic acid according to claim 1 with a DNA of nucleotides 1027-1965 from SEQ ED NO: 3.
3. Verfahren zur Auflösung von Zellgewebe und Freisetzung von darin enthaltenen Zellen oder Zellverbänden durch Inkubation des Zellgewebes mit einer neutralen Protease aus Clostridium histolyticum, welche codiert wird von3. Method for dissolving cell tissue and releasing cells or cell assemblies contained therein by incubating the cell tissue with a neutral protease from Clostridium histolyticum, which is encoded by
a) einer DNA der Nukleotide 1027-1965 aus SEQ ID NO: 3 oder einer hierzu komplementären DNA,a) a DNA of nucleotides 1027-1965 from SEQ ID NO: 3 or a complementary DNA,
b) Nukleinsäuren, die mit einer DNA der Nukleotide 1027-1965 aus SEQ ID NO:3 hybridisieren,b) nucleic acids which hybridize with a DNA of nucleotides 1027-1965 from SEQ ID NO: 3,
c) Nukleinsäuren, die ohne die Degeneration des genetischen Codes mit einer der in a) oder b) genannten Nukleinsäuren hybridisieren würden,c) nucleic acids which would hybridize with one of the nucleic acids mentioned in a) or b) without the degeneration of the genetic code,
und das Produkt einer prokaryontischen oder eukaryontischen Expression einer exogenen Nukleinsäure ist, bis zur Freisetzung der Zellen oder Zellverbände im gewünschten Umfang und Abtrennung der Zellen oder der Zellverbände von den Zellgewebeanteilen. and the product is a prokaryotic or eukaryotic expression of an exogenous nucleic acid until the cells or cell assemblies are released to the desired extent and the cells or cell assemblies are separated from the cell tissue portions.
4. Verfahren nach Anspruch 3, dadurch gekennzeichnet, daß die Abtrennung über einen Dichtegradienten und Zentrifügation erfolgt.4. The method according to claim 3, characterized in that the separation takes place via a density gradient and centrifugation.
5. Verfahren nach den Ansprüchen 3 oder 4, dadurch gekennzeichnet, daß als Gewebe Pankreasgewebe verwendet wird, die isolierten Zellen Inselzellen und Zellverbände Inseln sind.5. The method according to claims 3 or 4, characterized in that pancreatic tissue is used as tissue, the isolated cells are islet cells and cell groups islets.
6. Verfahren nach Anspruch 3 bis 5, dadurch gekennzeichnet, daß als Zellgewebe Leber-, Haut-, Nabelschnur-, Endothel-, Knochen-, Muskel-, Herz-, Ovarial-, Uterus-, Fett- oder Plazentagewebe verwendet wird.6. The method according to claim 3 to 5, characterized in that liver, skin, umbilical cord, endothelium, bone, muscle, heart, ovarian, uterine, fat or placenta tissue is used as cell tissue.
7. Verfahren nach den Ansprüchen 3 bis 5, dadurch gekennzeichnet, daß als Zellgewebe Tumorgewebe verwendet wird.7. The method according to claims 3 to 5, characterized in that tumor tissue is used as cell tissue.
8. Verwendung einer neutralen Protease aus Clostridium histolyticum, welche codiert wird von8. Use of a neutral protease from Clostridium histolyticum, which is encoded by
a) einer DNA der Nukleotide 1027-1965 aus SEQ ID NO: 3 oder einer hierzu komplementären DNA,a) a DNA of nucleotides 1027-1965 from SEQ ID NO: 3 or a complementary DNA,
b) Nukleinsäuren, die mit einer DNA der Nukleotide 1027-1965 aus SEQ ID NO: 3 hybridisieren,b) nucleic acids which hybridize with a DNA of nucleotides 1027-1965 from SEQ ID NO: 3,
c) Nukleinsäuren, die ohne die Degeneration des genetischen Codes, mit einer der in a) oder b) genannten Nukleinsäuren hybridisieren würden,c) nucleic acids which would hybridize with one of the nucleic acids mentioned in a) or b) without the degeneration of the genetic code,
und das Produkt einer prokaryontischen oder eukaryontischen Expression einer exogenen Nukleinsäure ist, zur Auflösung von Zellgewebe und Freisetzung von darin enthaltenen Zellen oder Zellverbänden.and the product is a prokaryotic or eukaryotic expression of an exogenous nucleic acid, for the dissolution of cell tissue and the release of cells or cell assemblies contained therein.
9. Verfahren zur Herstellung eines Polypeptids, welches die Eigenschaften einer neutralen Protease aus Clostridium histolyticum hat, durch Expression einer exogenen Nukleinsäure in prokaryontischen und eukaryontischen Wirtszellen und Isolierung des gewünschten Polypeptids, wobei die Nukleinsäure für ein Protein mit der Aktivität der neutralen Protease aus Clostridium histolyticum codiert, dadurch gekennzeichnet, daß die Sequenz ausgewählt ist aus der Gruppe umfassend a) eine DNA der Nukleotide 1027-1965 aus SEQ ED NO:3 oder eine hierzu komplementäre DNA,9. A method for producing a polypeptide which has the properties of a neutral protease from Clostridium histolyticum, by expression of an exogenous nucleic acid in prokaryotic and eukaryotic host cells and isolation of the desired polypeptide, the nucleic acid for a protein with the activity of the neutral protease from Clostridium histolyticum coded, characterized in that the sequence is selected from the group comprising a) a DNA of nucleotides 1027-1965 from SEQ ED NO: 3 or a complementary DNA,
b) Nukleinsäuren, die mit einer DNA der Nukleotide 1027-1965 aus SEQ ID NO.3 hybridisieren,b) nucleic acids which hybridize with a DNA of nucleotides 1027-1965 from SEQ ID NO.3,
c) Nukleinsäuren, die ohne die Degeneration des genetischen Codes mit einer der in a) oder b) genannten Nukleinsäuren hybridisieren würden.c) nucleic acids which would hybridize with one of the nucleic acids mentioned in a) or b) without the degeneration of the genetic code.
10. Verfahren nach Anspruch 9, dadurch gekennzeichnet, daß eine DNA der Nukleotide 1027-1965 aus SEQ ID NO.3 verwendet wird.10. The method according to claim 9, characterized in that a DNA of nucleotides 1027-1965 from SEQ ID NO.3 is used.
11. Verfahren nach den Ansprüchen 9 oder 10, dadurch gekennzeichnet, daß der Wirt E.coli ist.11. The method according to claims 9 or 10, characterized in that the host is E. coli.
12. Verfahren nach den Ansprüchen 9 oder 10, dadurch gekennzeichnet, daß die Wirtszelle eine Hefezelle oder eine Insektenzelle ist.12. The method according to claims 9 or 10, characterized in that the host cell is a yeast cell or an insect cell.
13. Biologisch funktionelles Plasmid oder viraler DNA- Vektor, der eine DNA nach Anspruch 1 oder 2 enthält.13. A biologically functional plasmid or viral DNA vector which contains a DNA according to claim 1 or 2.
14. Prokaryontische oder eukaryontische Wirtszelle, welche mit einem DNA- Vektor nach Anspruch 13 stabil transformiert oder transfiziert ist.14. Prokaryotic or eukaryotic host cell which is stably transformed or transfected with a DNA vector according to claim 13.
15. Polypeptid mit der Aktivität einer neutralen Protease, welches von einer DNA codiert wird, die von der DNA der Nukleotide 1027-1965 aus SEQ ID NO:3 verschieden ist, aber mit dieser DNA hybridisiert. 15. Polypeptide with the activity of a neutral protease, which is encoded by a DNA which is different from the DNA of nucleotides 1027-1965 from SEQ ID NO: 3, but hybridizes with this DNA.
EP95942186A 1994-12-22 1995-12-20 Recombinant proteinase from clostridium hystolyticum and the use thereof for isolating cells and cell groups Withdrawn EP0799316A1 (en)

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DE4445891A DE4445891A1 (en) 1994-12-22 1994-12-22 Recombinant proteinase from Clostridium histolyticum and its use for the isolation of cells and cell assemblies
PCT/EP1995/005054 WO1996019583A1 (en) 1994-12-22 1995-12-20 Recombinant proteinase from clostridium hystolyticum and the use thereof for isolating cells and cell groups

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DE4445891A1 (en) 1996-06-27
US5853976A (en) 1998-12-29
CA2208324A1 (en) 1996-06-27
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WO1996019583A1 (en) 1996-06-27
JP2953477B2 (en) 1999-09-27
MX9704586A (en) 1997-10-31

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