Molecular cloning, expression, crystallization and mutation of staphylokinase

Song Hou-Yan, Tang Qi-Qun, Zhang Xiao-Xuan, Yu Ming, Liang Dong-Cai, Zhan Chun-Hui, Wan Zhu-Li

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)49
Number of pages1
JournalFibrinolysis and Proteolysis
Volume11
Issue numberSUPPL. 3
StatePublished - 1997
Externally publishedYes

ASJC Scopus subject areas

  • Hematology

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    Hou-Yan, S., Qi-Qun, T., Xiao-Xuan, Z., Ming, Y., Dong-Cai, L., Chun-Hui, Z., & Zhu-Li, W. (1997). Molecular cloning, expression, crystallization and mutation of staphylokinase. Fibrinolysis and Proteolysis, 11(SUPPL. 3), 49.