Crystallographic Study On The Structure Of SjTrx And Its Complex With SjTGR From Schistosoma Japonicum

Schistosomiasis,widely spread in tropical and sub-tropical areas,greatly threatened human's health and affected people's normal life.This disease is also a worlds' public health problem.The species is the Schistosoma japonicum in our country.Schistosoma's survival needs effective mechanisms to maintain its redox balance in cells.Thioredoxin system?Trx system?exists in all kinds of creatures and this is also one of the most important antioxidant systems in schistosoma.Thioredoxin glutathione reductase?SjTGR?is the central enzyme and thioredoxin?SjTrx?is the substrate of electron transfer in Schistosoma japonicum redox system,which plays a key role in maintaining redox balance involve in driving reactions in cell.In this study,schistosoma japonicum thioredoxin?Sj Trx?was prepared and the reduced activity was measured.We also determined the crystal structure of SjTrx.Meantime,we have prepared the protein complex of SjTGR-SjTrx and the preliminary crystallographic study was carried out.The main contents conclude:?1?Recombinant wt SjTrx protease used for structure and function studies was prepared.Target gene wtSjTrx were cloned from cDNA library of S.japonicum and inserted into the plasmid of pET28 a successfully via One Step Cloning Kit;then the SjTrx protein were expressed induced by IPTG at about 1.0 of OD600 in E.coli BL21?DE3?.Subsequently,the protein was purified with Ni affinity chromatography and DEAE affinity chromatography,and pure wt Sj Trx of above 95% were gained.?2?Characterization and the biological activity of recombinant wtSjTrx protease.Insulin reduction experiment were proceed to test the reductive activity of wt Sj Trx.Interactions of SjTrx with homologous Sec-SjTGR were measured,which confirmed its biological activity.?3?Determination of the crystal structure of wtSjTrx.The condensed wtSjTrx in high purity served as crystalline material were crystallized to obtain high diffracted resolution protein crystals.X-ray diffraction data shows that the crystals at a resolution of 2.0? which showed the protease three-dimensional structure and the structural characteristics of the active sites.?4?Design and preparation of SjTGR^C596S and SjTrx^C37S mutant proteins.Through site-direct mutagenesis,single base mutant gene was obtanined and the mutant proteins of SjTGR^C596S and Sj Trx^C37S were expressed and prepared,respectively.?5?Preparation of SjTGR-SjTrx protein complex.At a ratio of 1:5,excess SjTrx protease was incubated with SjTGR in vitro for 24 h at 4?.Subsequently the sample was applied to Superdex 75 gel column for preparation and separation the complex of SjTGR-SjTrx.?6?Preliminary crystallographic study of the SjTGR-SjTrx complex.The protein complex was concentrated to 10 mg/mL and crystallization conditions were screened.After optimization,the crystal was diffracted by X-ray and a data set at resolution of 4.0 ? was collected.