Abstract
The baculovirus/Sf9 cell expression can be employed as a powerful system for producing large amounts of the human erythrocyte glucose transporter, GLUT1 heterologously In order to exploit the system further, it is necessary to develop a convenient method for demonstrating that the transporter expressed in insect cells is biologically active. To achieve this, we have expressed the human CLUT1 in insect cells and photolabelled the expressed protein with [$^3$H] cytochalasin B, a potent inhibitor of the human erythrocyte glucose transporter. Subsequently, the labelled proteins were analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Membranes labelled with [$^3$H] cytochalasln B in the presence of L-Glucose yielded a single sharp peak of labelling of apparent $M_r$ 45,000 on SDS/polyacrylamide gels. The mobility of this peak corresponded exactly to that of the band detected by anti-glucose transporter antibodies on Western blots of membranes prepared from insect cells infected with recombinant virus. In addition, the sharpness of the radioactive peak provides further evidence for the conclusion that the expressed protein is much less heavily and heterogeneously glycosylated than its erythrocyte counterpart. No peak of labelling was seen with the membranes prepared from non-infected Sf9 cells. Furthermore, the incorporation of label into this peak was completely inhibited by the presence of 500 mM-D-Glucose during tile photolabelling procedure, showing the stereoselectivity of the labelling. These evidences clearly show that human glucose transporter expressed in insect cells exhibits native-like biological activity, and that photolabelling with [$^3$H] cytochalasin B can be a convenient means for analysing the biological activity of the transport protein expressed in insect cells.