Prokaryotic Expression Vector Construction and Encoded Protein Preparation of a Candidate Gene HvAP2-260 in Barley Spike Development

Purpose To Construct the prokaryotic expression vector of candidate gene HvAP2-260 in barley young spike development, and to prepare its high purity encoding protein.

Methods The specific PCR primers with sequence recognized by enzyme NdeI and XhoI were used to amplified cDNA templates, from young spike of barley variety ‘Morex’ at jointing stage. Multi-stage recombination method and directed linking methods were employed to sequentially construct the recombinants PET-30a(+)-HvAP2-260 and pCZN1-HvAP2-260; then, heat shock was used to transfer the recombinants into competent cells of Escherichia coli strain Trans1-T1 and Arctic Express, respectively. By using E. coli transformed with pCZN1 empty vector and uninduced positive clones as controls, the expression differences of protein encoded by the gene HvAP2-260 under different treatments were analyzed, and the expressed proteins were purified by affinity chromatography.

Results The 924 bp coding sequence (CDS) of gene HvAP2-260 was cloned successfully, a prokaryotic expression vector pCZN1-HvAP2-260 was constructed. The protein encoded by the HvAP2-260 gene could be efficiently induced to express in the Arctic-Express strain cells, but the expressed proteins were an inclusion body. The expressed proteins could be effectively purified by using a Ni-IDA-Sepharose Cl-6B chromatography column.

Conclusion The CDS of the barley spike development gene HvAP2-260 has been successfully cloned, the prokaryotic expression vector has been constructed, and the high-purity proteins encoded by the gene have been prepared. The prepared proteins are useful for fellow-up studies, such as antibody preparation, functional verification and action mechanism elucidation of the protein encoded by gene HvAP2-260 gene.