Optimization Of The Fermentation Conditions, Purification And Scale-up Study Of Recombinant Staphylokinase

Staphlokinase(Sak) is a small extracellular protein secreted by numerous strains of staphylococcus aureus, many experiences indicated that Sak was a more protein and more fibrin-specific fibrinolytic agent. This paper reported high yield production, purification and scale-up study of recombinant staphylokinase in B.Subtilis DB2.Recombinant staphylokinase was secreted to fermentation broth in a soluble, active form from B.Subtilis DB2.Fermentation condition effected the expression of r-Sak .In this paper, we studied mainly the conditions of fermentation from some factors, such as inoculum Quantities, induced time ,pH, obtained time et al .The optical conditions was found: inoculum 4%,initial pH 7.2,induced time at A600nm 2.0-2.5,it recovered 4-5h for obtaining after 20% sucrose Was added to the culture. Under these conditions, the expression of r-Sak reached 250mg/L,the enzymatic activity reached 65,000RU/ml. Highly purified recombinant staphylokinase(over 98%)was obtained from fermentation supernate by chromatography, at 4℃,on CM-Sephyrose F.F and on S-100 sephycryl column. CM-Sephyrose F.F and S-100 sephycryl column was scale-up 10-fold,it possessed as results as the former. A purification can obtain gram quantities of highly purified recombinant(over 98%).The semifinished product was tested for requirements of biologics, these results suggested that the semifinished product possessed 98% purify and reached 57,000RU/mg specific activity, its endotoxin was down 5EU/mg and residual quantities of bacterial proteins was down 1%.R-Sak was analysed by reduced SDS-PAGE,IFE, Edman protein sequence analysis, the results suggested that r-Sak has a molecular weight of 15,500 dal, a PI of 8.3,a N-terminal fifteen amino acid sequence of S-S-S-F-D-K-G-K-Y-K-K-G-D-D-A-.