title: Cloning and high-level expression of plasminogen activator inhibitor-1 cDNA derived from human glomerular mesangial cells.
abstract: Translation reading frame of human plasminogen activator inhibitor-1 (PAI-1) cDNA was amplified from total RNA extracted from glomerular mesangial cells by using reverse-transcription polymerase chain reaction (RT-PCR) and inserted into plasmid pUC19 in a sense orientation. Sequencing results revealed that PAI-1 cDNA had new initial codon ATG, but no signal peptide sequences, and translation reading frame was in agreement with PAI-1 cDNA derived from human endothelial cells. PAI-1 cDNA determined by sequencing was inserted into prokaryotic expression plasmid pBV220, and recombinant plasmid pBV220-PAI-l which had high-level expression inEscherichia coli was obtained. Recombinant PAI-1 protein attained to approximately 45 % of total bacterial proteins. Western blotting showed that there was a specific band in the region of 43.0 ku. Latent recombinant PAI-1 with 97 % purity was obtained from inclusion bodies after denaturation, renaturation and purification by FPLC. Recombinant PAI-1 activated by treatment with 4.0 mol/L guanidinium-hydrochloride had significant inhibition activity to u-PA and shared bioactivity in common with natural PAI-1.
authors.affiliation: Department of Nephrology, General Hospital of PLA, 100853, Beijing, China.
authors.forename: D
authors.initials: D
authors.lastname: Yibin
authors.forename: C
authors.initials: C
authors.lastname: Xiangmei
authors.forename: L
authors.initials: L
authors.lastname: Hongjun
authors.forename: Y
authors.initials: Y
authors.lastname: Yizhou
authors.forename: X
authors.initials: X
authors.lastname: Qihe
authors.forename: Y
authors.initials: Y
authors.lastname: Lifang