Expression And Crystallization Of Recombinant Sulfiredoxin From Arabidopsis Thaliana

Sulfiredoxin?Srx?,as a member of the oxidoreductase family,catalyzes the reduction of the Cysteine sulfinic acid of peroxiredoxin in presence of energy?ATP?and organic sulfur compounds in the form of S-RH especially when the organic sulfur compound is a thiol compound?Cys-SH?.Srx is involved in antioxidant metabolism by reactivating peroxiredoxin.When peroxiredoxin is inhibited by sulfinic acid,sulfiredoxin reactivates peroxiredoxin to participate in antioxidant metabolism by reducing sulfinic acid.Due to the presence of ATP and Srx,these lower oxidation states of cysteine?disulfide?and higher oxidation states such as sulfinic acid?Cys-SOOH?are easily reversed.However,little is known about the role of Srx in oxidative stress in plants.The purpose of this project is to obtain the recombinant sulfiredoxin of Arabidopsis thaliana,to screen and optimize the growth conditions of protein crystals,and to lay the foundation for its structure and function analysis.In this study,the sulfiredoxin gene was cloned into the expressions vectors pET-22b?+?and pET-28a?+?and expressed in a bacterial expression system using strains of E.Coli BL21?DE3?.The target protein was purified by nickel affinity chromatography and gel filtration,and identified by SDS-PAGE,we obtained higher purity and homogeneous protein.Through the screening of crystal growth conditions,a diffraction protein Srx crystal was obtained at the solution composed with 0.8 M NaH₂PO₄/1.2 M KH₂PO₄ for acetate pH4.5.The diffraction resolution is 3.2???.