Research Conopsea GcGASA Protein And Mutant Expression, Purification, Refolding And Oxidative Antimicrobial Antioxidant Activity

Gibberellin-induced cysteine-rich proteins (GIP) are found to be involved in many important physiological processes in plants, such as root growth, stem elongation, fruit maturation, and so on. Although little useful information is available concerning its secondary and tertiary structure, sequence alignment of amino acids shows that all members of this family share12conserved cysteine residues arranged in the order of CX3CX3CX8CX3CX2CCX2CXCX11CPCX12C, among which CXXC is usually presumed to possess redox activity.Methods:The heterogeneous gene of Gymnadnia conopsea (designated gcgasa) has been expressed in Escherichia coli BL21(DE3) using pET-28a(+) as the expression plasmid, meanwhile, mutant SS-GcGASA was successfully produced by Site-directed Mutagenesis. Subsequently, we carried out denaturation and oxidative refolding experiments on pET-28(a)-GcGASA protein and mutants. Following the purification and identification of target protein28a-GcGASA and mutant by gel filtration and SDS-PAGE, we investigated its antimicrobial activity and antioxidative activity to eliminate peroxide and DPPH in vitro and in vivo.Results:Sequencing data sugguested that the target gene has been successfully inserted into the expression vector pET28a SDS-PAGE analysis revealed that the heterogenous was expressed mainly in the form of inclusion body. Subsequently, we carried out denaturation and oxidative refolding experiments on pET28a-GcGASA and mutant. The studies on antioxidative activity revealed that the resultant oxidatively refolded protein pET28a-GcGASA had the capability to eliminate DPPH and H2O2radical, but its mutant did not. This phenomenon indicated that pET28a-GcGASA thereby performed antioxidative activity. The studies on antibacterial activities suggested that the variant GcGASA-SS exhibited stronger antifungal activity against Fusarium.G than wt GcGASA.