Prokaryotic Expression And Purification Of Gcthionin And Its Mutants And The Antifugal Activity Test

Plant defensin is an important component of the innate immune system. Theyusually contain45~54amino acid residues with the molecular weight of4~5kDa andthe isoelectric point (PI) of9. Plant defensin exhibits various biological activities,including a wide spectrum of antifungal activity, antibacterial activity in some cases,enzyme activity inhibitory function, as well as the inhibition on the growth of cancercells.Gcthionin is a novel plant defensin from Gymanadenia conopsea, a traditionalChinese herb. The gcthionin was screened from Gymnadenia cDNA library in ourprevious work. Gcthionin contains45amino acids, where the amino acids at site7andsite8are missing when compared with other family member. Apart from the conservedGly20, Gly32, Gly36and Arg39, other amino acids differ from one species to otherspecies. Swiss-model analysis revealed that unlike other family member, althoughthere occur eight Cys residues in Gcthionin, only3pairs of disulphide bonds can beformed.In this paper, pET-32(a)-DP-Gcthionin recombinant plasmid was constructed.Subsequently, several mutants were also constructed using site-directed mutagenesisPCR method. Following the protein expression and purification using Ni2+affinitychromatography, the fusion protein Trx-Gcthionin was acid-cleavaged and thendetected by Tricine-SDS-PAGE. Additionally, antifungal activity was also assayed inthis work.The mutants T10F, R31G mutant, I37F∕L38R mutants, and mutants I42wereconfirmed to be successfully constructed by sequencing them. Tricine-SDS-PAGEshowed that the target protein Gcthionin could be released following the treatment of50%formic acid or under the condition with pH=1.2. Antifungal assay revealed thatthe mycelia growth of Fusarium graminearum could be effectively inhibited with thepresence of wild type and mutant fusion. Furthermore, the inhibition activity is linearlydependent on the concentration. In addition, it was also found that both Gcthionin andthe synthesized peptide could inhibit the germination of Fusarium graminearum conidia. Further work needs to be done for the purpose to gain deeper understandingon the antifungal mechanism.