Expression and Activity of Tissue-type Plasminogen Activator Mutant Reteplase with Deletion of PAI-1 Binding SitesChinese Full Text
LI Fei;LI Zhong-pei;ZHAN Zhi;WANG Dong-xue;ZHU Mei-cai1;The Central Hospital Of TaiAN Clinical laboratory;Air Force General Hospital Clinical laboratory center;
Abstract: Objective: To construct a prokaryotic expression vector for tissue-type plasminogen activator mutantreteplase with deletion of a PAI-1 binding site, and valuate its biological activity by enzyme kinetics analysis. Methods: The recombinant plasmid pBV220-t-PA was transformed into E.coli for expression after DNA sequencing. The recombinant mutant t-PA inclusion body was purified with gel filtration, and then was purified with erythrina trypsin affinity chromatography after refolding. Results: The mutant t-PA encoding sequence was confirmed by DNA sequencing. The expression product was about 30% of whole lysis protein. After purified with gel filtration, the protein purity was more than 95%. The specific activity of mutant t-PA with deletion of PAI-1 binding site was 4.2×105IU/mg. This protein was not inhibited by PAI-1. The Kmwas 0.3298 μmol·L-1and Vmaxof mutant t-PA was 0.0476 μmol·min-1· g-1. Conclusion: Recombinant mutant t-PA with deletion of PAI-1 binding site prepared from E.coli could resist the inhibitions of PAI-1, and harbor a better biological activity.
- DOI：
10.13241/j.cnki.pmb.2014.16.008
- Series：
- Subject：
- Classification Code：
R440
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