Fermentation, purification and identification of recombinant RGD-Hirudin.
- Author:
Wei MO
1
;
Yan-Ling ZHANG
;
Long-Sheng WANG
;
Xin-Ying YANG
;
Hou-Yan SONG
Author Information
1. The Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, Shanghai 200032, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Fermentation;
Hirudins;
biosynthesis;
genetics;
isolation & purification;
pharmacology;
Male;
Molecular Weight;
Pichia;
genetics;
Platelet Aggregation Inhibitors;
pharmacology;
Rats;
Rats, Sprague-Dawley;
Recombinant Proteins;
biosynthesis;
isolation & purification;
pharmacology
- From:
Chinese Journal of Biotechnology
2004;20(1):126-129
- CountryChina
- Language:Chinese
-
Abstract:
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.
