Cloning and Expression of cDNA for Thrombin-like Enzyme
from Agkistrodon halys Pallas Snake Venom

PAN Hua, ZHOU Yuan-Cong, YANG Guan-Zhen and WU Xiang-Fu^*
( Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China )

Abstract 　　Total RNAs were extracted from the venom gland of the Agkistrodon halys Pallas snake. The thrombin-like enzyme gene was amplified by RT-PCR, cloned, and its nucleotide sequences have been determined. The enzyme called pallase cDNA encodes 708 nucleotides, namely 236 amino acids. Based on the homology, the catalytic residues and disulfide bridges of pallase were deduced as follows： catalytic residues, His⁴¹, Asp⁸⁶和Ser¹⁸²； and disulfide bridges, Cys⁷-Cys¹³⁹, Cys²⁶-Cys⁴², Cys⁷⁴-Cys²³⁴, Cys¹¹⁸-Cys¹⁸⁸, Cys¹⁵⁰-Cys¹⁶⁷和Cys¹⁷⁸-Cys²⁰³. The pallase expression plasmids under the control of the T7 promoter was constructed and the pallase was expressed in E.coli .
Key Words　　Agkistrodon halys Pallas; thrombin-like enzyme gene; clone; sequence analysis; expression in E.coli

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