Cloning and high-level expression of plasminogen activator inhibitor-1 cDNA derived from human glomerular mesangial cells
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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.
Keywordshuman plasminogen activator inhibitor-1 molecular cloning recombinant bioactivity
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