Sarkar H K, Thorens B, Lodish H F, Kaback H R
Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110.
Proc Natl Acad Sci U S A. 1988 Aug;85(15):5463-7. doi: 10.1073/pnas.85.15.5463.
The gene encoding the human erythrocyte glucose transporter, cloned from HepG2 hepatoma cells, was expressed in Escherichia coli by introducing a prokaryote-type ribosome binding site, subcloning the gene into the T7 promoter/T7 polymerase expression system, and transforming a strain that is defective in glucose transport. Cells bearing plasmids with the transporter gene take up 2-deoxy-D-glucose and D-glucose, unlike cells bearing plasmids without the transporter gene. Moreover, 2-deoxy-D-glucose uptake is inhibited by unlabeled D-glucose, cytochalasin B, or mercuric chloride but not by L-glucose. The glucose transport protein is inserted into the membrane of E. coli, as evidenced by immunoblotting experiments with two site-directed polyclonal antibodies, one directed against the COOH terminus of the glucose transporter and the other directed against a synthetic peptide containing amino acid residues 225-238. As detected with both antibodies, the protein migrates with apparent molecular mass of 34 kDa in sodium dodecyl sulfate/12% polyacrylamide, a size similar to that of the unglycosylated glucose-transport protein synthesized in vitro.
从肝癌细胞HepG2中克隆出的编码人红细胞葡萄糖转运蛋白的基因，通过引入原核生物型核糖体结合位点、将该基因亚克隆到T7启动子/T7聚合酶表达系统中，并转化葡萄糖转运缺陷型菌株，从而在大肠杆菌中得以表达。与携带不含转运蛋白基因质粒的细胞不同，携带含有转运蛋白基因质粒的细胞能够摄取2-脱氧-D-葡萄糖和D-葡萄糖。此外，未标记的D-葡萄糖、细胞松弛素B或氯化汞可抑制2-脱氧-D-葡萄糖的摄取，但L-葡萄糖无此作用。用两种位点特异性多克隆抗体进行免疫印迹实验证明，葡萄糖转运蛋白插入到了大肠杆菌膜中，一种抗体针对葡萄糖转运蛋白的COOH末端，另一种针对含氨基酸残基225 - 238的合成肽。用这两种抗体检测时，该蛋白在十二烷基硫酸钠/12%聚丙烯酰胺凝胶中迁移时的表观分子量为34 kDa，与体外合成的未糖基化葡萄糖转运蛋白的大小相似。
