High level expression of recombinant plasminogen activator inhibitor-1 in Escherichia coli and generation of its mutants involving Asp125, Glu128 and Glu130.
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.
Sui
G C
GC
Department of Biochemistry and Molecular Biology, College of Life Sciences, Peking University, Beijing, P.R. China.
Sun
H
H
Zhang
M
M
Hu
M H
MH
D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli; D006801:Humans; D016297:Mutagenesis, Site-Directed; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors
D018076:DNA, Complementary; D017395:Plasminogen Activator Inhibitor 1; D011993:Recombinant Fusion Proteins; D014568:Urokinase-Type Plasminogen Activator