高亲和力重组人硫氧还蛋白还原酶的原核表达、纯化和初步活性鉴定

Prokaryotic expression,purification and identification of the preliminary activity of high affinity recombinant human thioredoxin reductase

目的:分离纯化高亲和力的人硫氧还蛋白还原酶(TrxR)蛋白,完成初步的活性鉴定。方法:优化重组人TrxR蛋白的原核表达条件,再通过亲和层析法分离纯化得到高纯度的TrxR重组蛋白。采用5,5′-二硫双(2-硝基苯甲酸)(DNTB)法比较重组人TrxR蛋白、商业化鼠肝提取TrxR蛋白以及重组人TrxR-ΔC蛋白的酶活力,并用TrxR特异性抑制剂金诺芬(Auranofin)分别检测其对三种蛋白的抑制效率。结果:优化了重组人TrxR蛋白的原核表达条件,得到高纯度目的蛋白。在8个测量时间点(120~330 s),TrxR的吸光度>pET-TRS TER>TrxR-ΔC(P<0.05)。TrxR和pET-TRS TER的抑制率随抑制剂浓度的减小而减小;TrxR-ΔC蛋白在抑制剂浓度0.00000477μmol/mL的活性低于其他10个浓度(0.01950~5.00000μmol/mL)(P<0.05);抑制剂在0.0000191μmol/mL浓度时蛋白活性低于抑制剂浓度为5.00000μmol/mL(P<0.05)。结论:获得高纯度、高活力的重组人TrxR蛋白,证明C端硒半胱氨酸是TrxR蛋白发挥活性的重要氨基酸。

Objective:To purify high affinity recombinant human thioredoxin reductase(TrxR)and identify its preliminary activity.Methods:The prokaryotic expression of recombinant human TrxR protein was initially optimized,and then the recombinant protein was isolated and purified by affinity chromatography.5,5′-Dithiobis(2-nitrobenzoic)acid(DNTB)method was used to compare the activity of recombinant TrxR protein,commercially purified TrxR protein from rat liver and human TrxR-ΔC protein.Finally,the inhibition efficiency of the three proteins specific to TrxR was detected by Auranofin.Results:The prokaryotic expression conditions of recombinant TrxR protein were optimized,and the high purity target protein was obtained.The absorbance of TrxR>pET-TRS TER>TrxR-ΔC was obtained in the 8 measuring time points(ranging from 120 to 330 seconds)(P<0.05).The inhibition rate of TrxR and pET-TRS TER was decreased with reduced inhibitor concentration.TrxR-ΔC protein activity was lower at inhibitor concentration of 0.00000477μmol/mL than that at ten other concentrations(0.01950μmol/mL-5.00000μmol/mL)(P<0.05),and the protein activity was lower at inhibitor dose of 0.0000191μmol/mL than that at 5.00000μmol/mL(P<0.05).Conclusion:The recombinant TrxR protein with high purity and high activity was obtained.Our findings demonstrates that C-terminal selenocysteine should be an important amino acid in the activity of TrxR protein.