Preparation and activity analysis of RGD-mSAK (K130T, K135R) / 生物工程学报
Chinese Journal of Biotechnology
; (12): 456-460, 2005.
Article
in Zh
| WPRIM
| ID: wpr-305251
Responsible library:
WPRO
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.
Full text:
1
Index:
WPRIM
Main subject:
Oligopeptides
/
Pharmacology
/
Recombinant Proteins
/
Metalloendopeptidases
/
Molecular Sequence Data
/
Platelet Aggregation Inhibitors
/
Base Sequence
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Protein Engineering
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Platelet Aggregation
/
Escherichia coli
Limits:
Animals
Language:
Zh
Journal:
Chinese Journal of Biotechnology
Year:
2005
Type:
Article