Baculovirus-mediated expression of the Na+/glucose cotransporter in Sf9 cells.
We have used baculovirus (AcNPV) to express the Na+/glucose cotransporter protein in cultured Sf9 cells. We constructed a baculovirus transfer vector containing the cDNA for the rabbit intestinal Na+/glucose cotransporter (SGLT1) under the control of the polyhedrin gene promoter. Recombinant baculovirus was obtained by cotransfection of SF9 cells with wild-type AcNPV DNA and the transfer vector. Recombinant virus was identified by Southern blotting and then purified. Recombinant infected Sf9 cells expressed a protein which was recognized by anti-peptide antibodies raised to sequences of the cloned Na+/glucose cotransporter. This protein migrated with a molecular mass of 55 kD by SDS-PAGE, similar to the in vitro translation product of SGLT1. An identical protein was metabolically labeled with [35S]methionine. Cells which synthesized the transport protein showed Na(+)-dependent alpha MeGlc transport. Micromolar phlorizin inhibited transport. Uninfected and wild-type virus infected Sf9 cells did not have Na(+)-dependent glucose transport. All transport protein migrated at 45% sucrose (w/w) by density gradient sedimentation, suggesting that the expressed transporter is membrane associated. We conclude that we have functionally expressed the rabbit intestinal Na+/glucose cotransporter in Sf9 cells. The transporter is not heavily glycosylated, and this is consistent with previous work showing that glycosylation is not necessary for function. We are poised to purify and characterize this protein from a structure-function perspective.
Smith
C D
CD
Department of Physiology and Medicine, UCLA School of Medicine 90024-1751.
Hirayama
B A
BA
Wright
E M
EM
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA; D007700:Kinetics; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis* / Q000235:genetics; D009036:Moths; D010695:Phlorhizin / Q000494:pharmacology; D010957:Plasmids; D011817:Rabbits
D009004:Monosaccharide Transport Proteins; D004247:DNA; D010695:Phlorhizin