Prokaryotic Expression The Palm Parameter Gcgasa Protein Spectroscopy And Functional Studies

Gibberellin-induced cysteine-rich proteins(GIP),which have N-terminal signal peptides with varying length and share 12 conserved cysteine residues at C-terminal domain, play many siginificant roles in plants. However,the function of GIP in vitro and structural information were unknown up to now.Methods: The heterogeneous gene of Gymnadnia conopsea(designated gcgasa) has been expressed in Escherichia coli BL21(DE3) using pET-32 (a) as the expression plasmid. Following the purification and identification of fusion protein Trx-GcGASA , we investigated its existing state by Native-PAGE, antimicrobial activity and the ability to eliminate peroxide in vitro. Additionally, the intrinsic fluorescence of Trx-GcGASA has also been studied in the absence and presence of oxidant, reductant and GndHCl by using steady-state fluorescence spectroscopic methods.Results: Native-PAGE results revealed that fusion protein simultaneously exist in the form of monomer and trimer. The studies on physiological functions suggested that Trx-GcGASA might cause the aggregation of Escherichia coli with the concentration of 10 uM, however, it was not able to eliminate H2O2 directly. The intrinsic fluorescence results revealed that (1) Tyrosine made the dominating contribution to the overall fluorescence emission spectra following excitation at different wavelengths varying from 250 nm to 280 nm. (2) The introduction of DTT greatly enhanced the fluorescence emission from Trp (3) The treatment of GSSG or peroxide gave rise to the decrease on fluorescence intensity for both Tyr and Trp. (4) The fusion protein could not be fully unfolded in 6 M GdnHCl withλmax < 350 nm. (5) The unfolding equilibrium curve showed that the expressed and purified fusion protein experienced a sudden increase in fluorescence intensity at 346.2 nm in the concentraion of GdnHCl varying from 2 M to 3 M with the Gibbs free energy of ~ 3.7 kJ.mol-1. In contrast, a contrary tendency was detected for the denatued and reduced protein, where a remarkbale increase was followed by a sudden decrease on fluorescence intensity from 1.3 M to 2.6 M GdnHCl. In addition, the aforementioned results would be helpful for the further studies on the physiological functions,unfolding, refolding process and strucure for this protein.