Expression And Purification Of Ecdysteroid-regulated Protein From Chinese Mitten Crab Eriocheir Sinensis In E.Coli

The Chinese mitten crab (Eriocheir sinensis), one of the important economic aquaculturespecies in China. The cultivation of Chinese mitten crab has become the most importantindustry in China. But with the development of Chinese mitten crab aquaculture, there aremany problems happening in the process such as the diseases which are caused by the virusesand bacteria and the phenomenons of precocial, small individual and germplasm degradation,all of these take a heavy toll of the cultivation of Chinese mitten crab and also become thebottleneck of the cultivation. After improve the condition of applied, and the output still notgood, the researchers have started to study from the viewpoint of molecular biology. Lots ofresearches have showed that ecdysone in the Chinese mitten crab takes part in the active ofmolting and development, and the ecdysteroid-regulated protein has been found to beinvolved in the cascade reaction. So the researches of ecdysteroid-regulated gene in Chinesemitten crab have a significant meaningful in solving the problems of cultivation. This studyhas try to research the cdysteroid-regulated gene from the viewpoint of molecular biology, tryto make a more particular knowledge of the molting in Chinese mitten crab, solve theproblems in cultivation and further promote the sustainable development of the breeding.The goal of this study was to clone, efficiently express and purify theecdysteroid-regulated protein (ERP) in the form of a GST fusion protein. First of all, we usethe sofeware to analyse the ecdysteroid-regulated gene, including cDNA sequence analyse,three-dimensional structure prediction and multiple sequence alignment. After that the maturepeptide-coding cDNA fragment was extracted from Chinese mitten crap (Eriocheir sinensis),and then after using PCR to obtain the open reading frame (ORF), a recombinant plasmiddesignated pGEX-4T-1-Esr was successfully generated and showed to efficiently express theERP fusion protein as determined by SDS-PAGE. The resulting expressed protein wassuccessfully purified by a combination of affinity and conventional chromatographic methodssuch as GST column, Mono Q column and Gel Filtration. After purification, the recombinantprotein showed the expected size of41kDa on SDS-PAGE gels which was further confirmedby mass spectrometry and western blotting. Purification of recombinant protein was achievedby Fast Protein Liquid Chromatography (FPLC). About2.4mg/l recombinant protein withpurity more than90%was obtained.This is the first time we obtain the pure of ERP fusion protein more than90%, in the future work we’ll do the research on its function.