Expression and functional characterization of chitribrisin, a thrombin-like enzyme, in the venom of the Chinese green pit viper (Trimeresurus albolabris).
In the present study, functionally active, recombinant chitribrisin, which is a thrombin-like enzyme in the venom of the Chinese green pit viper (Trimeresurus albolabris), was expressed and purified using a prokaryotic system. The fusion protein of chitribrisin, together with TrxA and 6x His via an E.coli expression vector pET-32a(+), was successfully expressed in E.coli BL21(DE3) cells. After the fusion protein was isolated and purified by chelated Ni(2+) resin and specifically cleaved by enterokinase, the recombinant chitribrisin showed a strong fibrinogenolytic activity against the alpha and beta chains of human plasminogen-free fibrinogen and weak fibrinogen clotting activity. In addition, multiple sequence alignment revealed that the expressed chitribrisin was homologous to GPV-TL1 and GPV-TL2 from the snake venom of T. albolabris from central Thailand in terms of the amino acid sequence identities. However, there were some differences in the amino acid sequences of the proteins from the same species from different geographical locations. The causes for the geographical variation in TELs in the same species remain to be investigated. Mutagenesis of chitribrisin should be performed in future studies to study the structural and functional relationship and to identify the critical residues responsible for the properties of the thrombin-like enzyme.
Lin
Yixin
Y
Chongqing Engineering Research Center of Bioactive Substance, Chongqing Key Laboratory of Animal Biology, College of Biological Science, Chongqing Normal University, Shapingba, Chongqing 400047, PR China.
Yu
Xiaodong
X
He
Qiyi
Q
Li
Heng
H
Li
Dehua
D
Song
Xixun
X
Wang
Yusheng
Y
Wen
Haoping
H
Deng
Huanhuan
H
Deng
Jiangyu
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003435:Crotalid Venoms / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011517:Prothrombin Time; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013917:Thrombin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017841:Trimeresurus / Q000235:genetics*
D003435:Crotalid Venoms; D011993:Recombinant Fusion Proteins; D005340:Fibrinogen; D013917:Thrombin; C541452:chitribrisin, Trimeresurus albolabris