Purification and characterization of the bacterial plasminogen activator staphylokinase secreted by a recombinant Bacillus subtilis.
A gene coding for the bacterial plasminogen activator staphylokinase (SAK) was cloned from Staphylococcus aureus bacteriophage 42D into an exoprotease reduced mutant strain of Bacillus subtilis (1). Yields of up to 50 mg SAK per litre of culture supernatant were obtained depending on the medium used. SAK purified by ion exchange chromatography and gel filtration had a specific activity of 16,000 units/mg protein. Isoelectric focusing of the purified SAK revealed heterogeneity with respect to the isoelectric points. Four different SAK proteins were identified among which the majority fraction had an IEP of 6.3 and a N-terminal amino acid sequence of NH2-Lys-Gly-Asp ... This N-terminus was 10 amino acids downstream of the expected signal peptide cleavage site beyond AA 27. It resulted most likely from a postsecretory proteolytic modification of the transiently appearing and correct processing product. In contrast to other plasminogen activators SAK was found to be resistant to proteolytic inactivation by plasmin.
Gerlach
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D
Zentralinstitut für Mikrobiologie und experimentelle Therapie, AdW der DDR, Jena.
Kraft
R
R
Behnke
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D
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D015153:Blotting, Western; D003001:Cloning, Molecular; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D007526:Isoelectric Point; D008666:Metalloendopeptidases / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009154:Mutation; D010960:Plasminogen Activators / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics
D003470:Culture Media; D010960:Plasminogen Activators; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus