Expression of the human erythrocyte glucose transporter in Escherichia coli.
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.
Sarkar
H K
HK
Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110.
Thorens
B
B
Lodish
H F
HF
Kaback
H R
HR
D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D003001:Cloning, Molecular; D003571:Cytochalasin B / Q000494:pharmacology; D003847:Deoxyglucose / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007118:Immunoassay; D008113:Liver Neoplasms; D008627:Mercuric Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D014407:Tumor Cells, Cultured
D009004:Monosaccharide Transport Proteins; D003571:Cytochalasin B; D008627:Mercuric Chloride; D003847:Deoxyglucose; D005947:Glucose