High level expression in Escherichia coli of isopenicillin N synthase genes from Flavobacterium and Streptomyces, and recovery of active enzyme from inclusion bodies

Orna Landman, Dov Shiffman, Yosef Av-Gay, Yair Aharonowitz, Gerald Cohen*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

A T7 promoter-based vector was used to express the isopenicillin N synthase (IPNS) genes of Flavobacterium sp. 12,154 and Streptomyces jumomjinensis in Escherichia coli. Most of the IPNS synthesized at 37°C, and representing some 22% and 51% of the total cell protein respectively, occured in an insoluble, enzymatically inactive form. Active IPNS was recovered in a rapid and simple two-step procedure in which the insoluble material was first denatured in 5 M urea and then refolded by passing the solubilized IPNS through a G-25 Sephadex sizing column. Further chromatography on DEAE-Sepharose resulted in highly active IPNS preparations. This procedure was found to be well suited for scaling up to produce large amounts of IPNS.

Original languageEnglish
Pages (from-to)239-244
Number of pages6
JournalFEMS Microbiology Letters
Volume84
Issue number3
DOIs
StatePublished - 1 Dec 1991

Keywords

  • Flavobacterium
  • IPNS
  • Isopenicillin N synthase
  • Streptomyces jumonjinensis
  • β-Lactam antibiotics

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