Refolding of a staphylokinase variant Y1-sak by reverse dilution

Jintian He, Gaizhen Wang, Ruiguang Xu, Jinlin Feng, Jinlong Wang, Huabo Su, Houyan Song

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

To develop more potent thrombolytic agents with fibrinolytic and antiplatelet aggregation activity, staphylokinase (Sak) variant Y1-Sak, a recombinant mutant of the Staphylococcus aureus protein Sak, was constructed. Y1-Sak formed an insoluble inclusion body when overexpressed in Escherichia coli strain JF1125. To obtain an optimized refolding process, dilution refolding was used to optimize refolding conditions. The results revealed that additive l-arginine and refolding temperature played critical roles in the refolding of Y1-Sak. Subsequently, two refolding methods, gel filtration and reverse dilution, were investigated to refold Y1-Sak. The results indicated that the fibrinolytic activity and recovery of Y1-Sak from gel filtration were lower than those from reverse dilution. Reverse dilution refolding successfully reduced the side reaction of refolding with the help of l-arginine, and the fibrinolytic activity and recovery of Y1-Sak were significantly improved. Functional analysis revealed that refolded Y1-Sak by reverse dilution possessed fibrinolytic and antiplatelet aggregation activities. Moreover, the immunogenicity of Y1-Sak was significantly reduced.

Original languageEnglish (US)
Pages (from-to)29-41
Number of pages13
JournalApplied Biochemistry and Biotechnology
Volume151
Issue number1
DOIs
StatePublished - Oct 1 2008
Externally publishedYes

Keywords

  • Antiplatelet aggregation
  • Gel filtration refolding
  • RGD motif
  • Refolding
  • Reverse dilution refolding
  • Staphylokinase

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology

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