High-level expression in Escherichia coli of a chemically synthesized gene for [Leu-28]echistatin

Zhong Ru Can, Jon H. Condra, Robert J. Gould, Robert A. Zivin, Carl D. Bennett, John W. Jacobs, Paul A. Friedman, Mark A. Polokoff

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

A gene (Ecs) encoding a platelet aggregation inhibitor, echistatin (Ecs), has been chemically synthesized. Met at position 28 of the native protein was replaced by Leu in the recombinant Ecs. To express this synthetic gene in Escherichia coli, an expression vector, pJC264, was constructed by inserting portions of the E. colicheB and cheY gene complex into the plasmid pUC13. High-level expression of the synthetic [ Leu-28] Ecs was achieved by its fusion with the E. coli cheY gene in the expression vector. Recombinant [Leu-28]Ecs was liberated from the fusion protein by CNBr cleavage at the Met inserted between the CheY protein and [Leu-28]Ecs. The recombinant [Leu-28]Ecs was purified to homogeneity by reverse-phase high-performance liquid chromatography. The refolded [Leu-28]Ecs was identical to native Ecs in inhibiting platelet aggregation, suggesting that Met at position 28 is not essential for the biological activity of this platelet aggregation inhibitor.

Original languageEnglish (US)
Pages (from-to)159-166
Number of pages8
JournalGene
Volume79
Issue number1
DOIs
StatePublished - Jun 30 1989

Keywords

  • plasmid vector
  • platelet aggregation inhibitor
  • protein folding
  • Recombinant DNA

ASJC Scopus subject areas

  • Genetics

Fingerprint Dive into the research topics of 'High-level expression in Escherichia coli of a chemically synthesized gene for [Leu-28]echistatin'. Together they form a unique fingerprint.

Cite this