Preparation and activity analysis of RGD-mSAK (K130T, K135R).
- Author:
Bao-An NING
1
;
Ru MA
;
Yu-Ling ZHENG
;
Zhi-Xian GAO
;
Bo SHEN
;
Yong-Qiang JIANG
Author Information
1. Institute of Microbiology and Epidemiology, Academy of Military Medical Science, Beijing 100071, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Base Sequence;
Escherichia coli;
genetics;
metabolism;
Guinea Pigs;
Metalloendopeptidases;
biosynthesis;
metabolism;
Molecular Sequence Data;
Mutant Proteins;
biosynthesis;
genetics;
Oligopeptides;
metabolism;
Platelet Aggregation;
drug effects;
Platelet Aggregation Inhibitors;
pharmacology;
Protein Engineering;
Recombinant Proteins;
biosynthesis;
genetics;
isolation & purification
- From:
Chinese Journal of Biotechnology
2005;21(3):456-460
- CountryChina
- Language:Chinese
-
Abstract:
In order to construct RGD-mSAK mutant with reduced immunogenicity, and identify its biological activity after purification, mSAK gene fragment was amplified by over-lapping extension PCR. Then the gene was inserted into the prokaryotic expression vector pBV220 with P(R)P(L) promoters after confirmed by DNA sequencing; the expression plasmid pBV220-RGD-mSAK was constructed, and then was transformed into E. coli. DH5alpha. After temperature induction, the mutant Staphylokinase was over-expressed and much of protein was in the supernate of lysate, which is over 50% of total protein in the host. The protein was isolated and purified in Q-Sepharose FF, Sephacryl S-200 and SP, high purity protein was obtained and its purity was over 98%. The thrombolysis activity of the RGD-mSAK protein is 1.68 x 10(5) u/mg by fibrin plate assay, which is slightly higher than that of the wild-type, and antiserum titers raised against this protein in guinea pigs were much lower than those of wild-type SAK, determined by ELISA. In anti-platelets aggregation assay in vitro, the RGD-mSAK protein has obvious inhibition activity of platelet aggregation in low concentration comparing to the control group and wild-type SAK group. So the RGD-mSAK protein is a low immunogenicity, bi-function molecular with both thrombolysis activity and anti-embolism activity. It provided the basis for further research of RGD-SAK.
