Invasive Species Compendium

Detailed coverage of invasive species threatening livelihoods and the environment worldwide

Abstract

Molecular cloning, sequence analysis and expression in Escherichia coli of the Spodoptera frugiperda protein phosphatase 2A.

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) Sf-9 cell culture and expressed in Escherichia coli. The full-length 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 pET30a-PP2A 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°C 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.