Expression and characterization of the human erythrocyte anion exchanger in a baculovirus/Sf-9 cell system.
The human erythrocyte anion-exchange protein (HAE1) has been expressed in insect Sf-9 cells using a recombinant baculovirus. We subcloned the full-length cDNA encoding HAE1 into the baculovirus expression vector pVL1392 and cotransfected Sf-9 cells with the recombinant vector and wild-type AcMNPV DNA to obtain recombinant baculovirus. The expressed protein was targeted to the Sf-9 plasma membrane at an apparent density of approximately 0.5 x 10(6) copies/cell as determined by quantitative autoradiography using an HAE1-specific monoclonal antibody. Unlike native HAE1, the expressed protein was not glycosylated. Transport studies with HAE1-recombinant-infected Sf-9 cells showed saturable [Km(Cl-) = 44 mM; Vmax(Cl-) = 48 mEq/liter of cell waters min] and H2DIDS-inhibitable (K(O.5) = 34 microM) 36Cl- uptake that was not present in uninfected cells. We also found that extracellular SO4(2-) reduced 36Cl- influx [K(0.5)((SO4)2-) = 26 mM], presumably through substrate competition as in erythrocytes. Finally, we observed that H2DIDS-inhibitable 36Cl- efflux was reduced by 77% in the nominal absence of a suitable counter-anion in the external solution (HCO3(-)-free, all-glucuronate medium), thereby providing strong evidence for an obligatory exchange mechanism. We conclude that there is high-level expression of + ++HAE1 functional activity in recombinant baculovirus-infected Sf-9 cells and that this system will prove useful for kinetic and structural analyses of the HAE1 protein.
Dale
W E
WE
Department of Medicine, Veterans Affairs Medical Center, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Textor
J A
JA
Mercer
R W
RW
Simchowitz
L
L
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D017136:Ion Transport; D007700:Kinetics; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D013431:Sulfates / Q000494:pharmacology
D001457:Anion Exchange Protein 1, Erythrocyte; D002712:Chlorides; D011994:Recombinant Proteins; D013431:Sulfates; C018564:dihydro-DIDS; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid