Molecular cloning, expression, crystallization and mutation of staphylokinase

The natural staphylokinase was purified from culture, determined the N and C terminal amino acid sequences, and designed a pair of PCR primers. Sak gene was successfully amplified by PCR using chromosome of the same strain as a template. The complete nucleotide sequence of Sak gene was determined in both orientation. The expression plasmid pSTE-Sak containing Sak gene and regulatory factors PR, PL promoters was constructed. Sak gene was over expressed in a special E. coli strain. The r-Sak with MW 15500, covered 50% of total bacterial protein and existed in an active soluble form. r-Sak was purified by ion-exchange. The final product with 98-99% of purity and 10'HU/mg of special activity was obtained 400-500 mg from a liter culture. The thrombolytic therapy of r-Sak for rabbit femoral artery thrombosis, intraocular hemorrhage and fibrin formation were successful.. The purified recombinant staphylokinase was fully active and readily crystallized against 1.2 M sodium citrate in 100 mM Tris-HCl buffer at pH 8.9 by hanging-drop method. Crystals of staphylokinase diffract to better than 2.2A resolution. The crystal belongs to the tetragonal space group p4,2]2 or its enantiomorph with unit-cell parameters a=b=67.5,c = 150.1 A. There are two molecules in the asymmetric unit. C terminal, N and C terminals deleted mutations were studied. The preliminary x-ray diffraction and structure/function of mutations data of our r-Sak were different from other reports.