Molecular cloning, sequence analysis and expression in Escherichia coli of the Spodoptera frugiperda protein phosphatase 2A
Author of the article:HAO ShaoDong**YANG BaoDongWANG JinZhong***ZHANG ZhiYong***ZHANG MinZhaoZHENG LinQingSUN ShuLi
Author's Workplace:(Beijing Key Laboratory of New Technology in Agricultural Application ,College of Plant Science and Technology,Beijing University of Agriculture, Beijing102206,China
Key Words:Spodoptera frugiperda, PP2A, molecular cloning, prokaryotic expression
Abstract:This study is aimed at establishing a method of PP2A activity assay for evaluating potential new pesticides in vitro. A Ser/Thr protein phosphatase 2A catalytic subunit gene was cloned from Spodoptera frugiperda (J.E. Smith) Sf9 cell culture and expressed in Escherichia coli. The fulllength cDNA sequence was 1 303 bp, encoding 309 amino acids with a predicted molecular weight of 35.46 ku and an isoelectric point of 5.37. This PP2A may mainly occur in cytoplasm without signal peptides and transmembrane structure. Multiple comparison analysis of S. frugiperda and other insects’ PP2A indicate that PP2A is highly conserved. PP2A of S. frugiperda, the target enzyme for selection of inhibitors, can be used for screening insecticides in activity assays. A prokaryotic expression vector pET30aPP2A was constructed and transfected into the E. coli BL21 (DE3) stain, establishing a prokaryotic expression system. This expression system expressed PP2A well at conditions of 16-30℃ with a 0.2-0.8 mol/L IPTG inducer. We dissolved the inclusion in urea solution and purified it using a Ni\|agarose column, obtaining purified PP2A which presented a single band after SDS\|PAGE, showing the purity was over 90%. These results lay a foundation for research into PP2A activity assay.