Efficient purification of selenoprotein thioredoxin reductase 1 by using chelating reagents to protect the affinity resins and rescue the enzyme activities

Abstract The application of 2’,5’-ADP Sepharose affinity chromatography is used to purify the recombinantly expressed selenoprotein thioredoxin reductases (TrxRs, TXNRDs). However, the affinity resin gradually loses its binding capacity, and the eluted fractions show low activities. The issues above might be attributed to either impairment of the ligands of the column by the released intercellular nucleases or inhibition of the enzyme by trace heavy metal impurities. In this study, chelating reagents are supplemented in crude samples and buffer, and the effects on enzyme purification are investigated. Our results show that 20 mM EDTA or EGTA fully protects/rescues TrxR1 from the impairment of heavy metal ions. More importantly, 20 mM EDTA improves the yield of TrxR1 from 50 % to >80 % after 2’,5’-ADP Sepharose affinity chromatography, therefore increasing the reproducibility of the 2’,5’-ADP Sepharose column. The purified enzyme shows an apparent 55-kDa subunit single band on a reducing SDS-PAGE gel and exhibits a specific activity >20 U/mg based on the classic DTNB reduction assay. This study would be helpful for the high-efficiency preparation of selenoproteins using NADPH as a cofactor and for the effective protection of ribonucleotide-based ligands via affinity chromatography.