[Cloning and secretory expression of staphylokinase in Streptomyces lividans].
OBJECTIVE
Construct a recombinant Streptomyces strain by which staphylokinase can be secretory expressed.


METHODS
With the total DNA extracted from strains of Staphylococcus aureus isolated in clinic as template DNA, one 730 bp DNA fragment including secretory signal peptide sequence and structure gene of staphylokinase was amplified by PCR. After cloning into plasmid pUC19 and sequencing, this fragment was confirmed to have the same nucleotide sequence as that of staphylokinase gene reported before. Then the fragment was inserted into Streptomyces plasmid pIJ459 following strong promoter. Recombinant plasmid pIJ459SAK was transformed into S. lividans TK54.


RESULTS
The 72 hours culture supernatant of the secretory-type genetic engineering strain that harbored pIJ459SAK was analyzed by 15% SDS-PAGE. One specific protein band with molecular weight about 16,000 can be seen after staining the gel with silver nitrate. The fibrinolytic activity of Staphylokinase can be also detected in the 72 hours culture supernatant in the fibrin-plate assay.


CONCLUSIONS
Staphylokinase can be successfully secreted into fermentation culture by the recombinant Streptomyces strain.
Cheng
Y
Y
Institute of Medicinal Biotechnology, CAMS and PUMC, Beijing 100050.
Li
Y
Y
Liu
B
B
Guo
L
L
D003001:Cloning, Molecular; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus