High level expression of recombinant plasminogen activator inhibitor-1 in Escherichia coli and generation of its mutants involving Asp125, Glu128 and Glu130.

Abstract

Plasminogen activator inhibitor-1 (PAI-1) cDNA was expressed in Escherichia coli (E. coli) with high efficiency using a heat-inducible vector. About 100 mg of recombinant PAI-1 (rPAI-1) could be obtained from 1 liter of bacteria culture. rPAI-1 in inclusion bodies was purified by pI precipitation and Sephadex G-75 chromatography. After treatment with 4 mol/L guanidinium chloride and dialysis, the largely inactive PAI-1 gained considerably in activity as judged by its reaction with low molecular weight u-PA (LMW-u-PA). Degenerated oligonucleotides containing ApaI site and mutations at Asp125, Glu128, Glu130 in PAI-1 cDNA were synthesized. To facilitate the introduction of mutations, an ApaI site was first generated in PAI-1 cDNA using one of the oligonucleotides. Taking advantage of the APaI site, thirteen PAI-1 mutants involving Asp125, Glu128 and Glu130 were produced with these oligonucleotides using PCR. Most of the PAI-1 mutants had a similar activity as compared to wild type PAI-1, while some of the triple-site mutants had completely lost their activity.