Activated Expression And Efficient Purification Of Mammalian Selenoprotein Thioredoxin Reductase 1 (TrxR1) In E. Coli

Mammalian cytosolic thioredoxin reductase 1(Trx R1)is a kind of selenoprotein withimportant roles in antioxidant defense and redox regulation,principally linked to functions of their main substrates thioredoxins(Trx).It is highly associated with occurrence and development of various diseases including tumor and inflammation.A deep understanding of the nature and function of Trx R1 is highly dependent on obtaining high quality of Trx R1.So far,expression and purification of mammalian selenoprotein face some problems such as low recovery rate and low enzyme activity.So this research focus on prokaryotic expression process and strategy for separation and purification of Trx R1.The main work includes three aspects as following:1.Optimizing of prokaryotic expression: By adding the riboflavin precursor vitamin B2(riboflavin)or adding divalent metal ions such as Mg2+,Mn2+ and Ca2+ to the growth medium to improve the prokaryotic expression of Trx R1.Concerning the problems of low yield of Trx R1 in shake flask culture and low enzyme activity which need to be improved.We found riboflavin can enhance Trx R1 expression in the concentration range of 0-1000 ?M.Especially when the concentration of riboflavin was 10 ?M,the effect of promoting expression was significant,the enzyme activity increased by 60%.Mg2+ can enhance Trx R1 expression in the concentration range of 0-300 ?M.Especially when the concentration of Mg2+was 200 ?M,the effect of promoting expression was significant,the enzyme activity was increased by 189%.Mn2+ can enhance Trx R1 expression in the concentration range of0-200 ?M.Especially when the concentration of Mg2+ was 100 ?M,the effect of promoting expression was significant,the enzyme activity increased by 26%.Ca2+ can enhance Trx R1 expression in the concentration range of 0-1000 ?M.Especially when the concentration of Ca2+ was 400 ?M,the effect of promoting expression was significant,the enzyme activity was increased by 44%.2.Improving the strategy of separation and purification: We used Sephacryl S-300 gel filtration to replace much more expensive Superdex G-200.Coupled with ADP Sepharose of the first step,we got the same purification result and reduced the cost of the purification matrix.In the phase of ADP Sepharose affinity chromatography,sample pretreatment was carried out by adding chelating agent EDTA to Trx R1 crude enzyme,thus protecting ADPligand on affinity chromatography matrix and effectively enhance affinity chromatography yield of Trx R1.When the concentration of EDTA was 20 m M,the yield was increased about33%.The stability and reproducibility of affinity chromatography column is well.In the purification process,we used Sephacryl S-300 gel filtration to replace Superdex G-200,which greatly reduced the cost.The final yield of Trx R1 was about 72%.SDS-PAGE electrophoretic result showed a single band,indicating purification result was well.3.Characterization the properties of Trx R1: We investigated the effect of different metal ions on Trx R1 enzyme activity and found many metal ions(such as Ca2+,Cd2+,Co2+,Fe3+and so on)inhibited the DTNB reduction activity of Trx R1 with concentration dependence.DTNB reduction activity of Trx R1 reduced rapidly with the increase of metal ion concentration.The inhibitory effect was greatly relieved during the purification process with metal ions chelated by EDTA in crude enzyme solution.In conclusion,the results of this study optimized the prokaryotic expression of Trx R1,improved the separation and purification strategy of Trx R1 and characterized the inhibitory effect of metal ions on Trx R1.It laied the foundation for the preparation of high quality Trx R1,offered assistance for research on properties of Trx R1 and provided a reference for the expression and purification of other selenoproteins.