Prokaryotic Expression and Purification of HC-Pro Protein of Papaya Leaf Distortion Mosaic Virus
Papaya leaf distortion mosaic virus(PLDMV,genus Potyvirus)is becoming a new major limiting factor for the economical production of papaya in China.Helper component-proteinase(HC-Pro)is a potyviral multi-functional protein involved in virus movement,replication,vector transmission,and RNA silencing.In order to ob-tain the purified protein of HC-Pro gene,this study successfully constructed the prokaryotic expression plasmid pET-30a-HCPro by codon optimization of HC-Pro gene sequence;the highest expression of HC-Pro in E.coli was achieved after induction with IPTG and was induced overnight at 0.5 mmol/L IPTG at 20 ℃;after fragmentation,SDS analysis of the expression of target proteins in whole bacteria,fragmented supernatants and precipitates showed that the recombinant HC-Pro protein was mainly expressed in the precipitates as insoluble inclusion bodies;further purification of the solubilized inclusion bodies by means of Ni-NTA chromatography columns resulted inHC-Pro re-combinant protein with a molecular weight size of approximately 54 kD at a concentration of500 mmol/L imidazole;western Blot analysis of the purified target protein showed a clear band at 54 kD,consistent with the expected size of the HC-Pro recombinant protein.In this study,PLDMV recombinant protein HC-Pro was successfully expressed and purified,which provided a theoretical basis for subsequent antibody preparation and investigation of its function.
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