[Expression and characterization of carboxylesterase A2 in E. coli].
The most commonly observed change that has been linked to resistance development is the increase in activity of carboxylesterases. The putative mechanism involves an overproduction of this enzyme for the sequestration and the hydroxylation of various organophosphate and carbamate insecticides. Carboxylesterases A2 cDNA was amplified from Culex quinquefasciatus by RT-PCR and sequenced consequently. Target gene was inserted into pET-28a to create prokaryotic expression plasmid pET-EstA2. When pET-EstA2 was transformed into E. coli BL21, the recombinant was induced by IPTG. A pure recombinant protein was obtained by affinity purification. Compared with carboxylesterase A2 purified from Culex quinquefasciatus, carboxylesterase A2, purified from the product of the transgenic of E. coli, has the same Km, but the Vm was higher than that of it, which shows that carboxylesterase A2, purified from the product of E. coli by affinity, is purer than that from Culex quinquefasciatus. The study on the expression and characterization of carboxylesterase A2 in E. coli is more useful for its future application.
Huang
Jing
J
The State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, the Chinese Academy of Sciences, Beijing 100080, China.
Qiao
Chuan Ling
CL
Zhao
Qiao
Q
Wang
Peng
P
D000818:Animals; D043182:Carboxylesterase / Q000235:genetics / Q000378:metabolism*; D003465:Culex / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D008957:Models, Genetic; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D043182:Carboxylesterase