cDNA cloning and high-level expression of a thrombin-like enzyme from Agkistrodon acutus venom.
Agkistrodon acutus (Guenther), a poisonous snake species of the family of Crotalidae, is mainly found south of the Yellow River in China. The main symptom of this poison is massive hemorrhage, in which thrombin-like enzymes (TLEs) in the venom play an important role. TLEs are abundant, especially in the venom of A. acutus. We isolated the total RNA from the venom gland tissue of A. acutus and amplified the cDNAs of the TLEs using reverse transcription-polymerase chain reaction (RT-PCR). The cDNAs were cloned into vector pThioHis B and were expressed as fusion proteins in the form of inclusion bodies, which accounted for nearly 50% of the total cell proteins. The inclusion bodies were washed, dissolved, refolded and purified by affinity chromatography. The purity was higher than 97%, as indicated by capillary zone electrophoresis. The renatured recombinant enzyme exhibited arginine esterase activity, as tested by the BAEE method, and also showed a fibrinogen cleavage effect, as detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This method provides a fast and convenient system for studying the structure-function relationships in TLE isoenzymes, and also a practical way for mass production of TLEs in the pharmaceutical industry.
Zha
X D
XD
School of Life Science, University of Science and Technology of China, Hefei, PR China.
Ren
B
B
Liu
J
J
Xu
K S
KS
D017836:Agkistrodon; D000595:Amino Acid Sequence; D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry; D003435:Crotalid Venoms / Q000201:enzymology*; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D005260:Female; D005340:Fibrinogen / Q000378:metabolism; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013917:Thrombin / Q000235:genetics*
C031442:Agkistrodon venoms; D003435:Crotalid Venoms; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D005340:Fibrinogen; C019724:arginine esterase; D002265:Carboxylic Ester Hydrolases; D013917:Thrombin