Cloning,
Expression and Purification of Gussurobin, A Thrombin-like Enzyme from the Snake Venom of Gloydius
ussuriensis
YANG Qing, HU Xue-Jun, XU Xiao-Ming, AN
Li-Jia*, YUAN Xiao-Dong１, SU Zhi-Guo2
( Bioengineering Institute, Dalian University of Technology, Dalian 116012,
China；1Takara Biotech Dalian Co. Ltd., Dalian 116600, China; 2The
State Key Laboratory of Biochemical Engineering, Institute of Chemical Metallurgy, the Chinese Academy of
Sciences, Beijing 100080, China )
JANSON Jan-Christer
( Biosurface Science Center, Biomedical Center 577, 75 123 Uppsala,
Sweden )
Abstract Total RNAs
were extracted from the venom gland of the snake Gloydius ussuriensis.
The cDNA of gussurobin, a thrombin-like enzyme from Gloydius ussuriensis, was cloned and amplified by RT-PCR.
Assay of the nucleotide sequence of the cDNA allowed postulation of the
complete amino acid sequence for Gloydius ussuriensis, Chinese Viperdae. Its amino acid
sequence exhibits significant homology with that of other snake thrombin-like
enzymes. The cDNA of gussurobin was inserted into the vector pPIC 9K and
expressed successfully in Pichia pastoris, strain GS115. The recombinant gussurobin was separated and
purified from 500 ml culture and showed one band on SDS-PAGE.
Key words thrombin-like enzyme; gussurobin; cDNA sequence;
expression; purification
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