Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-01-23
2008-08-05
Zeman, Robert A. (Department: 1645)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S183000, C435S200000, C435S212000, C435S219000, C435S320100, C424S094640, C424S243100, C424S190100, C530S300000, C530S350000
Reexamination Certificate
active
07407789
ABSTRACT:
The present invention relates to the biotechnology field, more particularly, to novel recombinant staphylokinase (RGD/KGD-Sak) derivatives and the preparation the thereof. The derivatives, have a low polymerizing ability, low immunogenicity and a bifunctionality of thrombolytics and anticoagulant. Based on the trimensional structural analysis of the monomer and dimer of recombinant staphylokinases and their biochemical properties, we designed two novel bifunctional staphylokinase molecular structures. Mutant genes were constructed by PCR site-directed mutagenesis, which were then recombined with a prokaryotic vector and used to transformE. coli. Engineered strains with a high expression level were selected by screening and propagated by fermentation, followed by disruption of the cells, centrifugation to collect inclusion bodies, renaturation, and purification of RGD/KGD-SAK through a two-step method. After lyophilized, the polymerizing ability and immunogenicity of the products decreased significantly. The derivatives can not only activate fibrinogen to lyse thrombus, but also significantly inhibit the platelet aggregation induced by ADP, suggesting that they have the bifunctionality of thrombolytics and anticoagulant.
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Song Gang
Song Houyan
Cook Alex McFarron Manzo Cummings & Mehler, Ltd.
Fudan University
Tongue Lakia J
Zeman Robert A.
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