[Preparation and activity analysis of RGD-mSAK (K130T, K135R)].
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.
Ning
Bao-An
BA
Institute of Microbiology and Epidemiology, Academy of Military Medical Science, Beijing 100071, China.
Ma
Ru
R
Zheng
Yu-Ling
YL
Gao
Zhi-Xian
ZX
Shen
Bo
B
Jiang
Yong-Qiang
YQ
D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006168:Guinea Pigs; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000378:metabolism; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000096:biosynthesis* / Q000235:genetics; D009842:Oligopeptides / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
D050505:Mutant Proteins; D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D011994:Recombinant Proteins; C047981:arginyl-glycyl-aspartic acid; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus