Expression, purification and identification of gibberellin-induced cysteine-rich protein of Gymanadenia conopsea

Abstract

According to the known partial cDNA sequence of gibberellin-induced cysteine-rich protein from Gymnadnig conopsea,the primers bearing restriction enzyme site of EcoR I and HindⅢ were designed for amplifying the full-length of ORF and the signal peptide-truncated fragment of gcgasa gene. Two fragments with the length of 319 and 238 bp were obtained and then cloned into the plasmid pET-32(a). Following the transformation into Escherichia coli BL21(DE3) , the fusion proteins were observed at approximately 26.0 and 25.2 kD with the induction of 1 mmol/L IPTG. The results of SDS-PAGE and transmission electron micrograph of ultrathin section revealed that the presence of signal peptide gave rise to the formation of inclusion body, which was located in periplasmic space, however, the absence of signal peptide greatly enhanced the solubility of the target protein, and the expressed soluble protein was further purified by Ni2+-NTA affinity chromatography and gel filtration methods.