Cloning, Expression, and Renaturation Studies of Reteplase

  • Zhao, Youchun (State Key Laboratory of Microbial Technology, Shandong University) ;
  • Ge, Wang (State Key Laboratory of Microbial Technology, Shandong University) ;
  • Kong, Young (State Key Laboratory of Microbial Technology, Shandong University) ;
  • Zhang, Changkai (State Key Laboratory of Microbial Technology, Shandong University)
  • 발행 : 2003.12.01

초록

Recombinant human tissue plasminogen activator deletion mutein (Reteplase) is a clinically promising thrombolytic drug. Reteplase cDNA was subcloned into a bacteria expression system, and the resultant recombinant was biologically characterized. The Reteplase was expressed in Escherichia coli as an inclusion body, and the downstream processes of the Reteplase inclusion body included denaturation, renaturation, and purification. A protein disulfide isomerase (PDI) was used to assist the refolding of Reteplase, and it was found to increase the refolding rate from less than 2% to more than 20%. The refolded Reteplase was purified through two chromatography steps, including lysine-coupled agarose affinity chromatography and then CM-sepharose cation-exchange chomatography. The purity of r-PA was analyzed by Western bolt analysis, and N-terminal amino acid and amino acid composition analyses confirmed the end-product. Reteplase showed higher thrombolytic potency in an animal thrombus model.

키워드

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