Overexpression of pseudomonas fluorescens lipase in escherichia coli

Abstract

The lipase gene which previously cloned from Pseudomonas fluorescens SIK Wl in our laboratory was subcloned into expression vector, pTTQ 19 and expressed in E. coli JM 103. The pTTQ 19 vector contains a polylinker/lacZα flanked by the strong tac promoter and γγB transcription terminator, and contains the $lacl^q$ allele of the lac regressor gene to ensure optimal regulation. The lipase gene generated from pJH 92 (pUC 19 derivative harboring the lipase gene) was inserted into the polylinker region of this expression cassette, to give transnational fusion within the lacZα coding region. The resultant plasmid was named as pTTy 2. When the culture of E. coli harboring pTTY 2 was induced with IPTG (final concentration 0.5 mM), high levels of the lipase were expressed from pTTY 2. The lipase of the total cell protein and was found exclusively in cytoplasm as an insoluble form. Aggregates of the insoluble form, termed inclusion bodies, were shown to be highly refratile under phase contrast microscope. When the inclusion bodies which had no biological activity were solubilized with 8 M urea and then gradually dialyzed, lipolytic activity was recovered. And an exceeding 10 mg of crude lipase could be obtained in 100 ml culture.