[Expression of snake venom thrombin-like enzyme calobin in Pichia pastoris].
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.
Yuan
Shengling
S
Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.
Wang
Peng
P
Tao
Haoxia
H
Zhan
Dewen
D
Wang
Yanchun
Y
Wang
Lingchun
L
Liu
Chunjie
C
Zhang
Zhaoshan
Z
D017836:Agkistrodon; D000818:Animals; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics; D013917:Thrombin / Q000096:biosynthesis* / Q000235:genetics; D014757:Viper Venoms / Q000201:enzymology*
D011994:Recombinant Proteins; D014757:Viper Venoms; D012697:Serine Endopeptidases; C104841:calobin; D013917:Thrombin