Overproduction of staphylokinase in Escherichia coli and its characterization.
A recombinant plasmid which directs the overproduction in Escherichia coli of staphylokinase from Staphylococcus aureus has been constructed by placing the staphylokinase gene, sak, under the control of bacteriophage lambda PR promoter in the plasmid. When an E. coli strain having the plasmid was induced, the staphylokinase activity in the periplasmic fraction increased about 60-fold and the 15.5-kDa protein corresponding to the mature form reached about 25% of the periplasmic proteins. At the same time the 18.5-kDa protein corresponding to the precursor form was accumulated in the membrane fraction, showing that the processing and translocation of the sak gene product were restricted during high level of its synthesis. By using this strain, the mature staphylokinase has been easily purified to near homogeneity. The purification steps consisted of extraction of the periplasmic proteins by osmotic shock and CM-cellulose column chromatography. Two species of staphylokinase were identified after CM-cellulose column chromatography. Although their isoelectric points and NH2-terminal amino acid sequences were different, their specific activities were almost equal. These results strongly suggest that the NH2-terminal portion of staphylokinase is not important for its activity.
D002852:Chromatography, Ion Exchange; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000096:biosynthesis* / Q000235:genetics; D004792:Enzyme Precursors / Q000096:biosynthesis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008666:Metalloendopeptidases; D009876:Operon; D010957:Plasmids; D013347:Subcellular Fractions / Q000201:enzymology; D013696:Temperature
D004274:DNA, Recombinant; D004792:Enzyme Precursors; D010450:Endopeptidases; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus