人细胞质硫氧还蛋白(hTrx1)在抗氧化和氧化还原调控中起重要作用.如果静脉注射重组hTrx1, 动物抗氧化能力将增高. 近年来, 随着人们对氧化还原调控的关注, hTrx1需求不断增加. 为了快速获得高纯度重组hTrx1, N末端亲和标签, 如组氨酸标签(His-tag)和谷胱甘肽S转移酶标签(GST-tag), 被用于hTrx1亲和纯化.带N末端标签的hTrx1融合蛋白在实验中用的越来越多.但N末端延长是否会影响hTrx1特性尚不清楚. 我们构建与优化了hTrx1原核表达质粒, 在大肠杆菌中高效表达了含天然N末端、带His-tag或带 GST-tag的3种重组hTrx1纯化蛋白在SDS-PAGE上呈现1条带, 对应的分子量分别为12 kD、17 kD及38 kD. 在无氧化剂存在时, 它们催化胰岛素还原的能力不分仲伯. 当有H2O2存在时, 天然N末端hTrx1通过形成可逆二聚体, 对H2O2表现出较强的耐受性; 而N末端亲和标签有干扰二聚体形成, 使hTrx1对H2O2耐受性降低的作用, 其中GST-tag干扰作用明显大于His-tag. 此外,体内重要的氧化还原对GSH/GSSG, 有增进hTrx1及其还原酶催化NADPH氧化的作用, N末端亲和标签可明显扩大GSH/GSSG的这种作用. 我们分析了N末端亲和标签对hTrx1活性影响的可能机理.

Human cytosolic thioredoxin (hTrx1) plays an important role in antioxidation and redox regulation. Intravenous administration of recombinant hTrx1 has been shown to enhance its antioxidant capacity. As proteins involved the redox-regulation, including hTrx1, has received attentions in recent years, the N-terminal affinity tags, such as His-tag and glutathione S-transferase (GST)tag, have been used in hTrx1 affinity chromatography to facilitate fast purification of highpurity hTrx1, and the tags were kept attached in some applications. However, whether the N-terminal extensions will affect the properties of hTrx1 remained unclear. In this study, we over-expressed the hTrx1 with or without an N-terminal His or GST-tag in E. coli. The purified proteins displayed similar activities in catalyzing the reduction of insulin. Native hTrx1 formed reversible dimers and was resistant to H2O2. The GST or His-tagged hTrx1 was more sensitive to H2O2 treatment with a decreased dimer formation, especially for the hTrx1 with a GST-tag. In addition,GSH/GSSG,and important redox pair in vivo,may increase the rate of NADPH oxidation catalyzed by Trx system,which was significantly amplified by the N-terminal affinity tags.The possible mechanisms underlying the effects of the tags on hTrx1 activ were discussed.