The role of thioredoxin and disulfide isomerase in the expression of the snake venom thrombin-like enzyme calobin in Escherichia coli BL21 (DE3).
Thrombin-like enzymes (TLEs) are studied widely because of their therapeutic potential in myocardial infarction and thrombotic diseases. But they are always produced in Escherichia coli BL21 (DE3) as inclusion bodies because they are single chain Cys-rich proteins. In this work, coexpressing of thioredoxin (TrxA) largely increased the solubility of calobin, one of the TLEs from korea viper Agkistrodon caliginosus, but soluble calobin-T had poor enzyme activity. Disulfide isomerase (DsbC) was introduced into the host cell and coexpressed with calobin in the presence of TrxA, the result was that both the solubility and enzyme activity of calobin-TD were higher than those of calobin-T. Although recombinant calobins exhibited the enzyme of hydrolyzing the fibrinogen, they lost clotting activity to the substrate. Recombinant calobin-TD remained poor amidolytic activity, the effects of divalent metal cations and various inhibitors on this activity were similar to those of native calobin nevertheless, suggesting that calobin-TD exhibited the characteristics of serine protease especially of trypsin-like serine protease.
Beijing Institute of Biotechnology, 20 Dong Dajie Street, Fengtai District, Beijing 100071, China.
Shengling
S
Yuan
Haiqing
H
Duan
Chunjie
C
Liu
Xiuli
X
Liu
Taotao
T
Liu
Haoxia
H
Tao
Zhaoshan
Z
Zhang
D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D012910:Snake Venoms / Q000201:enzymology / Q000378:metabolism*; D012911:Snakes / Q000378:metabolism*; D013696:Temperature; D013879:Thioredoxins / Q000378:metabolism*
D011994:Recombinant Proteins; D012910:Snake Venoms; D013879:Thioredoxins; D012697:Serine Endopeptidases; C104841:calobin; D019704:Protein Disulfide-Isomerases