title: [Preparation and activity analysis of RGD-mSAK (K130T, K135R)].
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.
authors.affiliation: Institute of Microbiology and Epidemiology, Academy of Military Medical Science, Beijing 100071, China.
authors.forename: Bao-An
authors.initials: BA
authors.lastname: Ning
authors.forename: Ru
authors.initials: R
authors.lastname: Ma
authors.forename: Yu-Ling
authors.initials: YL
authors.lastname: Zheng
authors.forename: Zhi-Xian
authors.initials: ZX
authors.lastname: Gao
authors.forename: Bo
authors.initials: B
authors.lastname: Shen
authors.forename: Yong-Qiang
authors.initials: YQ
authors.lastname: Jiang
mesh_terms: 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*
chemical_list: D050505:Mutant Proteins; D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D011994:Recombinant Proteins; C047981:arginyl-glycyl-aspartic acid; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus