Abstract：Objective: To construct the prokaryotic expression vector for gcgasa gene encoding Gibberellin-induced cysteine-rich protein, and to express the gene in E.coli BL21(DE3). Material: Gymanadenia Conopsea R.Br. Methods: The primers bearing restriction enzyme site of EcoR I and Hind III were designed and were ultilized to amplify the full-length of ORF and the signal peptide-truncated fragment. The target fragments were then cloned into the pET-32(a) vector to produce the recombinant plasmids pET-32(a)/gcgasa and pET-32(a)/gcgasa. After they were identified by DNA sequencing, the recombinants were transformed into E.coli BL21(DE3) to express the fusion protein with the induction of IPTG. SDS-PAGE, Western blotting, as well as Transmission Electron Micrograph of ultrathin section were done to identify the expression and location of heterogenous protein. Following the solubility analysis, the expressed soluble protein was further purified by Ni2+-NTA affinity chromatography and gel filtration methods. Results: DNA sequencing showed that the recombinant plasmids have been successfully constructed. SDS-PAGE and Western blotting analysis 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. In the latter case, further purification was achieved using Ni2+-NTA and Sephadex G-75 gel filtration column. Conculsion: It was for the first time that the soluble gcgasa protein was successfully obtained from E. coli system with high efficiency. This study will be very fundamental and essential for further investigation on the stucture and function of gcgasa.
