Expression of snake venom thrombin-like enzyme calobin in Pichia pastoris.
- Author:
Shengling YUAN
1
;
Peng WANG
;
Haoxia TAO
;
Dewen ZHAN
;
Yanchun WANG
;
Lingchun WANG
;
Chunjie LIU
;
Zhaoshan ZHANG
Author Information
1. Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
- Publication Type:Journal Article
- MeSH:
Agkistrodon;
Animals;
Pichia;
genetics;
metabolism;
Recombinant Proteins;
biosynthesis;
genetics;
Serine Endopeptidases;
biosynthesis;
genetics;
Thrombin;
biosynthesis;
genetics;
Viper Venoms;
enzymology
- From:
Chinese Journal of Biotechnology
2009;25(4):526-532
- CountryChina
- Language:Chinese
-
Abstract:
Thrombin-like enzymes (TLEs) are studied widely because of their therapeutic potential in myocardial infarction and thrombotic diseases. We synthesized the DNA fragment encoding thrombin-like enzyme calobin from Agkistrodon caliginosus (Korean Viper) venom by fusion PCR and expressed it in Pichia pastoris. After induction by 0.5% methanol for 48 h, the expression level of recombinant calobin reached 3.5 g/L in medium. The recombinant calobin was purified by Q-Sepharose Fast Flow ion-exchange chromatography and Sephacryl-S-100 gel filtration chromatography. Purified sample had a molecular weight of 32 kD shown in SDS-PAGE. It hydrolyzed fibrinogen and formed a light white hydrolysis circle in fibrinogen plate. SDS-PAGE analysis showed that recombinant calobin cleaved Aalpha-chain of fibrinogen specifically, and produced an appropriately 40 kD new band. However, we failed to find its fibrin-clot formation activity.
