Recombinant proteinase from clostridium histolyticum and its use

Details Recombinant proteinase from clostridium histolyticum and its use Recombinant proteinase from clostridium histolyticum and its use

1997-08-26

1998-12-29

Carlson, Karen Cochrane

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

435 691, 4353201, 4352523, 435325, 435183, 4352542, 4352528, 435219, 536 231, C12Q 100, C12P 2106, C07H 1700, C12N 900

Patent

active

058539760

DESCRIPTION:

BRIEF SUMMARY
The invention concerns a recombinant proteinase (neutral protease, NP) from Clostridium histolyticum and its use for isolating cells and groups of cells.
Proteolytic enzymes from Clostridium histolyticum are used to digest tissues and to isolate individual cells or groups of cells (e.g. islets) (islets: Sutton et al., Transplantation 42 (1986) 689-691; liver: Quibel et al., Anal. Biochem. 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 collagenase types are known from Clostridium histolyticum (M. F. French et al., J. Protein Chemistry 11 (1992) 83-97).
In addition to various isoforms of type I and type II collagenase, a neutral protease (NP) from Clostridium histolyticum is also known the activity optimum of which is in the neutral pH range and which cleaves casein as well as 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 regarded as being necessary as an auxiliary enzyme in the digestion of various tissues (bones: Hefley et al., Exp. Cell Res. 149, 1983, 227-236; pancreas: Wolters et al., Diabetologica 35, 1992, 735-742). NP has a molecular weight of ca. 35 kD (SDS gel electrophoresis).
In order to use neutral protease to isolate cells and groups of cells on a large scale it is necessary to provide the neutral protease in a reproducible quality and in large amounts. This is possible by recombinant production processes.
Therefore the object of the present invention was to provided nucleic acids which code for proteins with the activity of the neutral protease from Clostridium histolyticum as well as 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 which is characterized in that it is selected from the group comprising complementary thereto, SEQ ID NO:3, hybridize with one of the nucleic acids mentioned in a) or b).
A nucleic acid is preferred of nucleotides 1027-1965 from SEQ ID NO:3. Nucleic acids are also suitable which, compared to this nucleic acid, are shortened or extended preferably at the 5' end by for example ca. 60 nucleotides. An extended nucleic acid of nucleotides 970-1965 is particularly preferred which corresponds to a proform of the protease. Shortened nucleic acids correspond to proteolytically, preferably autoproteolytically, processed proteins.
The activity of neutral protease is known to a person skilled in the art and described by Mandle et al. (1953) Sparrow and McQuade (1973) and Hefley (1987). Neutral protease cleaves casein as well as denatured collagen.
Hybridization within the sense of the invention is understood as a hybridization under the usual stringent conditions familiar to a person skilled in the art as they are for example stated by J. Sambrook in Molecular Cloning, Cold Spring Harbor Laboratory (1989) and B. D. Hames, S. G. Higgins, Nucleic Acid Hybridization--A practical approach (1985), IRL-Press, Oxford, England. Usually the standard protocols are used for the hybridization which are described in these publications.
"Stringent conditions" are preferably understood as a hybridization in 6.0.times.SSC at about 45.degree. C. with a subsequent washing step at 2.0.times.SSC at 50.degree. C. In order to adjust the stringency the salt concentration can for example be selected in the washing step of 2.0.times.SSC at 50.degree. C. for low stringency to 0.2.times.SSC at 50.degree. C. for high stringency. In addition the temperature of the washing step can be set between room temperature (22.degree. C., low stringency) to about 65.degree. C. (high stringency). The stringent hybridization conditions are preferably selected such that at least a homology of 75% preferably of 90% is obtained in the amino acid sequence.
A DNA or RNA is suitable within the sense of the invention as a nucle

REFERENCES:
Wolters, et al., Diabetologia, vol. 35, 1992, pp. 735-742, "An analysis of the role of collagenase and protease in the enzymatic dissociation of the rat pancrease for islet isolation".
Hefley, et al., Experimental Cell Research, vol. 149, 1983, pp. 227-236, "Enzymatic isolation of cells from neonatal calvaria using two purified enzymes from Clostridium histolyticum".
Wetmore, et al., Molecular Microbiology, vol. 6, 1991, pp. 1593-1604, "The role of the pro-sequence in the production and secretion of the thermolysin-like neutral protease form Bacillus cereus".
Meinhardt, et al., Appl. Microbiol. Biotechnol., vol. 41, 1994, pp. 344-351, "Cloning and sequencing of the leuC and npr genes and a putative spo IV gene from Bacillus megaterium DSM319".

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