科研之友
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摘要
Staphylokinase(STA),a M(r) 18000 protein produced by Staphylococcus aureus has profibrinolytic properties (Lack CH, Nature 161, 559-560, 1948), but its potential for thrombolytic therapy has not been thoroughly investigated. Therefore we have elaborated procedures for the large scale production of natural (STAN) and recombinant (STAR) staphylokinase. A strain of Staphylococcus aureus (strain no. 23), selected from 100 consecutive cultures at our Bacteriology Laboratory, was found to secrete up to 300 mug staphylokinase per litre culture broth within 18h. The material, purified by batch adsorption on SP-Sephadex and chromatography on insolubilised active site-blocked plasmin with a yield of approximately 40 mug per 1, migrated as a single band on SDS gel electrophoresis with M(r) almost-equal-to 16 500 and had NH2-terminal sequence Lys-Gly-Asp-Asp-Ala-. Restriction enzyme digested genomic DNA from this strain was hybridised with a degenerate 14-mer deoxyoligonucleotide encoding the NH2-terminal sequence, hybridising fragments were isolated and ligated into pUC19, competent E. coli cells were transformed, recombinant clones isolated and culture medium assayed for STA activity. A clone (subclone 159-2) transformed with recombinant pUC 19 containing a 2.9 kb insert obtained by HindIII restriction enzyme digestion was found to secrete STAR without further manipulation (up to 20 mg/l into the culture broth and 30 mg/l into the periplasmic space). The material, purified by chromatography on insolubilised active-site blocked plasmin with a yield of 50 and 25%, respectively, contained three STAR variants with NH2-terminal sequence Ser-Ser-Ser-Phe-Asp-Lys-Gly-Lys-Tyr-Lys-Lys-Gly-Asp-Asp-Ala- (STA-M), with Gly-Lys-Tyr-Lys-Lys-Gly-Asp-Asp-Ala- (STA-DELTA6) and with Lys-Gly-Asp-Asp-Ala- (STA-DELTA10). Chromatography on CM-Sephadex yielded two peaks, STA-CM-I containing STA-DELTA10 and STA-CM-II containing STA-M and STA-DELTA6. The specific activities of STAN, of STA-DELTA10 obtained from STAR and of mixtures of STA-M and STA-DELTA6 from STAR were indistinguishable by clot lysis and plasminogen coupled chromogenic substrate assays. Thus, highly purified preparations of natural (STAN) and recombinant (STAR) staphylokinase can be obtained by simple and straightforward techniques in sufficiently large amounts to allow detailed investigation of their biochemical and thrombolytic properties.
- 出版日期1992-10
