Sherratt P J, Pulford D J, Harrison D J, Green T, Hayes J D
Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, U.K.
Biochem J. 1997 Sep 15;326 ( Pt 3)(Pt 3):837-46. doi: 10.1042/bj3260837.
The cDNA encoding human glutathione S-transferase (GST) T1 has been expressed as two recombinant forms in Escherichia coli that could be purified by affinity chromatography on either IgG-Sepharose or nickel-agarose; one form of the transferase was synthesized from the pALP 1 expression vector as a Staphylococcus aureus protein A fusion, whereas the other form was synthesized from the pET-20b expression vector as a C-terminal polyhistidine-tagged recombinant. The yields of the two purified recombinant proteins from E. coli cultures were approx. 15 mg/l for the protein A fusion and 25 mg/l for the C-terminal polyhistidine-tagged GST T1-1. The purified recombinant proteins were catalytically active, although the protein A fusion was typically only 5-30% as active as the histidine-tagged GST. Both recombinant forms could catalyse the conjugation of glutathione with the model substrates 1,2-epoxy-3-(4'-nitrophenoxy)propane,4-nitrobenzyl chloride and 4-nitrophenethyl bromide but were inactive towards 1-chloro-2,4-dinitrobenzene, ethacrynic acid and 1-menaphthyl sulphate. Recombinant human GST T1-1 was found to exhibit glutathione peroxidase activity and could catalyse the reduction of cumene hydroperoxide. In addition, recombinant human GST T1-1 was found to conjugate glutathione with dichloromethane, a pulmonary and hepatic carcinogen in the mouse. Immunoblotting with antibodies raised against different transferase isoenzymes showed that GST T1-1 is expressed in a large number of human organs in a tissue-specific fashion that differs from the pattern of expression of classes Alpha, Mu and Pi GST. Most significantly, GST T1-1 was found in only low levels in human pulmonary soluble extract of cells, suggesting that in man the lung has little capacity to activate the volatile dichloromethane.
编码人谷胱甘肽S-转移酶（GST）T1的cDNA已在大肠杆菌中以两种重组形式表达，这两种形式可通过在IgG-琼脂糖或镍-琼脂糖上进行亲和层析来纯化；一种形式的转移酶由pALP 1表达载体合成，为金黄色葡萄球菌蛋白A融合蛋白，而另一种形式由pET-20b表达载体合成，为C末端多组氨酸标签重组蛋白。从大肠杆菌培养物中获得的两种纯化重组蛋白的产量分别约为：蛋白A融合蛋白15 mg/L，C末端多组氨酸标签的GST T1-1为25 mg/L。纯化后的重组蛋白具有催化活性，尽管蛋白A融合蛋白的活性通常仅为组氨酸标签的GST的5%-30%。两种重组形式均可催化谷胱甘肽与模型底物1,2-环氧-3-(4'-硝基苯氧基)丙烷、4-硝基苄基氯和4-硝基苯乙基溴的结合反应，但对1-氯-2,4-二硝基苯、依他尼酸和1-萘基硫酸酯无活性。发现重组人GST T1-1具有谷胱甘肽过氧化物酶活性，可催化氢过氧化异丙苯的还原反应。此外，发现重组人GST T1-1可使谷胱甘肽与二氯甲烷结合，二氯甲烷是小鼠的一种肺和肝致癌物。用针对不同转移酶同工酶产生的抗体进行免疫印迹分析表明，GST T1-1在大量人体器官中以组织特异性方式表达，这与α、μ和π类GST的表达模式不同。最显著的是，在人肺细胞可溶性提取物中仅发现低水平的GST T1-1，这表明在人类中，肺激活挥发性二氯甲烷的能力较弱。
