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| The Prokaryotic Expression of an Anti-coagulant Protein of EH |
| ZHANG Chao1, GONG Wei1, GUO Ying-ying1, SUN Wei-guo2, YAO Min3, YU Ai-ping1 |
1. Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850, China;
2. The 309 hospital, PLA, Beijing 100091, China;
3. The Medical Library of the Chinese PLA, Beijing 100039, China |
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Abstract Objective: EH is a derivative of hirudin with three amino acids (EPR) at the amino terminal of hirudin. The expression of EH in yeast has long time of production and low efficiency of expression. To improve the production efficiency of EH, the soluble expression of EH in E. coli was studied. Method: Three recombinant prokaryotic expression engineering bacteria, BL21 (DE3)-pET-24-eh, BL21(DE3)-pET-22-eh, BL21(plySs)-pET-22-eh were constructed. The three engineering bacteria were cultured and EH expression was induced by IPTG. Results: The results of SDS-PAGE and Western blot showed that EH was expressed in intracellular soluble pattern in all three engineering bacteria, and the expression level is better in BL21(DE3)-pET-24-eh. Then in BL21(DE3)-pET-24-eh, the induction temperature, the induction time, the concentration of IPTG and the density of bacteria at induction time were optimized. The optimized condition is as follows: induction at 37 ℃ for 6 h, IPTG concentration is 0.4μmol/L, the bacteria density at induction time is OD600 = 1. Finally, the specific anticoagulant activity of purified EH protein (the purity is 96.93%) and the cleavage product of EH by FXa were determined. Conclusion: The results indicated that the intact EH has no anticoagulant activity, and after cleavage with FXa, EH released anticoagulant activity of hirudin. Therefore, the prokaryotic expression system of EH, and EH were constructed which can be expressed in an intracellular soluble pattern. These results will support the subsequent fermentation process and the ultimate industrialization of EH.
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Received: 09 September 2014
Published: 25 December 2014
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