Expression, Structure And Characterization Of Wheat Thioredoxin And Glutaredoxin

Glutaredoxin(GRX) and thiredoxin(TRX) are small ubiquitous oxidoreductases, and they have wide application prospects in food, medicine and cosmetic industry.In this thesis, wheat glutaredoxin(WGRX) and thioredoxin(WTRX) were expressed in E.coli system and purified by affinity chromatography and size exclusion chromatography. Then, the enzymatic properties of WGRX and WTRX were studied. Then the interactions of WGRX and WTRX and the effects of them on the flour quality were initially explored. Also, the structure of WGRX was solved. The main contents and results are as follows:(1) Construction of recombinant expression plasmids and sequences ananlysis. The total RNA was extracted from wheat etiolated seedling of yannong 19 and then the DNA fragments were obtained by reverse transcription and PCR and then ligated to pMD-19 T vector. The results of sequences analysis showed that WGRX belongs to the GRX C1 subgroup and WTRX belongs to the TRX h subgroup Ⅱ. Liagating the fragments to the pET-30 b vector and the E.coli expression system were constructed successfully.(2) Expression and purification of WGRX and WTRX. The evaluation of induction conditions(temperature, IPTG concentration and time) in E.coli was accomplished.The optimal expression condition for WGRX were determined that the induction temperature was 16 ?C, the concentration of IPTG was 0.05 mmol/L and the induction time was 12 h. The optimal expression condition for WTRX were determined that the induction temperature was 37 ?C, the concentration of IPTG was 0.4 mmol/L and the induction time was 12 h. The pure protein was obtained by affinity chromatography and size exclusion chromatography.(3) Characterization of WGRX and WTRX. The results showed that the optimal reaction pH for WGRX was 8.0 and temperature was 40 ?C and it had a specific activity of 0.2 U/mg. WGRX had a good stability when pH varied from 6-8 and tempetature varied from 20 ?C-60 ?C. The optimal reaction pH for WTRX was 7.0 at temperature was 30?C and it had a specific activity of 18 U/mg. WTRX has a good stability when pH varied from 5-11 and tempetature varied from 20 ?C-70 ?C. In suitable reaction system, WTRX may be reduced by WGRX and adding their reaction system to the flour at the same time could improve the development time of flour.(4) The crystallization and the structure determination of WGRX. The high quality of WGRX crystals were obtained by screening and optimization. The structure of WGRX was solved successfully by the method of molecular replacement. There were 5 monomers arranged in line per asymmetrical unit and the monomer conformed to the thioredoxin fold, which consisted of three β-strands flanked by five α-helixs. In the cryatal structure, the WGRX mainly existed as redeced state.