[Fermentation, purification and identification of recombinant RGD-Hirudin].
Recombinant RGD-Hirudin ( r-RGD-Hirudin ) has double functions: anti-thrombin activity and anti-platelet aggregation activity. To identify these functions, the expression plasmid, RGD-Hirudin-pPIC9K, was constructed by inserting cDNA of RGD-hirudin in yeast expression vector pPIC9K. The high expression clone was gained after screening. This clone was fermented for 3 days. The r-RGD-hirudin was secreted into the culture. It was ultra-filtrated from culture supernatant, then after gel filtration chromatography and anion exchange chromatography, the purified r-RGD-hirudin was gained. Its purity was larger than 97% and its specific activity was 12 000 ATU/mg. The yield per liter culture of purified r-RGD-hirudin was 1 g and overall recovery yield was more than 75% . The purified r-RGD-hirudin was identified by reductive SDS-PAGE, anti-thrombin activity assay, anti-platelet aggregation assay, LC/MS and isoelectrofocusing assay. It is proved that r-RGD-Hirudin is ramification of wt-Hirudin and it has anti-thrombin activity and anti-platelet aggregation activity.
Mo
Wei
W
The Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, Shanghai 200032, China.
Zhang
Yan-Ling
YL
Wang
Long-Sheng
LS
Yang
Xin-Ying
XY
Song
Hou-Yan
HY
D000818:Animals; D005285:Fermentation; D006629:Hirudins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology
D006629:Hirudins; D010975:Platelet Aggregation Inhibitors; C544959:RGD-hirudin; D011994:Recombinant Proteins