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.
Similar content being viewed by others
References
Sprenger, E.D., Kluft, C., Plasminogen activator inhibitor,Blood, 1987, 69:381.
Wong, A. P., Cortez, S.L., Baricos, W.H., Role of plasmin and gelatinase in extracellular matrix degradation by cultured rat mesangial cells,Am. J. Physiol., 1992, 263 (6 Pt2): F1112.
Reilly, T.M., Seetharam, R., Duke, J.L. et al., Purification and characterization of recombinant plasminogen activator inhibitor-1 fromEscherichia coli, J. Biol. Chem., 1990, 265:9570.
Sisk, W. P., Davis, G. L., Kingsley, D. et al., Highlevel synthesis of biologically active human plasminogen activator inhibitor type 1 (PAI-1) inEscherichia coli., Gene, 1990, 96:305.
Seetharam, R., Dwivedi, A., Duke, J.L. et al., Purification and characterization of active and latent forms of recombinant plasminogen activator inhibitor 1 produced inEscherichia coli, Biochemistry, 1992, 31: 9877.
Pannekoet, H., Veerman, H., Lambers, H. et al., Endothelial plasminogen activator inhibitor (PAI): a new member of the serpin gene family,The EMBO Journal, 1986, 5: 2539.
Sumbrook, J., Fritsch, E.F., Maniatis, T.,Molecular Cloning: Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratry Press, 1989.
Ny, T., Sawdey, M., Lawrence, D. et al., Cloning and sequence of a cDNA coding for the human β-migrating endothelialcell-type plasminogen activator inhibitor,Proc. Natl. Acad. Sci. USA, 1986, 83: 6776.
Ginsburg, D., Zeheb, R., Yang, A. Y. et al., cDNA cloning of human plasminogen activator-inhibitor from endothelial cells,J. Clin. Invest., 1986, 78: 1673.
Declerck, P.J., Demolm, M., Vaughan, D.E. et al., Identification of a conformationally distinct form of plasminogen activator inhibitor-1 acting as a noninhibitory substrate for tissue-type plasminogen activator,J. Biol. Chem., 1992, 267: 11698.
Reilly, T.M., Mousa, S.A., Seetharam, R. et al., Recombinant plasminogen activator inhibitor type I: a review of structural, functional, and biological aspects,Blood Coagul Fibrinolysis, 1994, 5: 73.
Author information
Authors and Affiliations
Additional information
Project supported in part by the National Natural Science Foundation of China (Grant No. 39421005) and Chinese Military Science Foundation.
Rights and permissions
About this article
Cite this article
Deng, Y., Chen, X., Liao, H. et al. Cloning and high-level expression of plasminogen activator inhibitor-1 cDNA derived from human glomerular mesangial cells. Sci. China Ser. C.-Life Sci. 41, 315–322 (1998). https://doi.org/10.1007/BF02895108
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF02895108