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 th e full-length cDNA encoding HAE1 into the baculovirus expression vecto r 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 densi ty of similar to 0.5 x 10(6) copies/cell as determined by quantitative autoradiography using an HAE1-specific monoclonal antibody. Unlike na tive HAE1, the expressed protein was not glycosylated. Transport studi es with HAE1-recombinant-infected Sf-9 cells showed saturable [K-m(Cl- ) = 44 V-max; (Cl-) = 48 mEq/liter of cell water . min] and H2DIDS-inh ibitable (K-0.5 = 34 mu M) Cl-36(-) uptake that was not present in uni nfected cells. We also found that extracellular SO42- reduced Cl-36(-) influx [K-0.5(SO42-) = 26 mM], presumably through substrate competiti on as in erythrocytes. Finally, we observed that H2DIDS-inhibitable Cl -36(-) efflux was reduced by 77% in the nominal absence of a suitable counter-anion in the external solution (HCO3--free, all-glucuronate me dium), thereby providing strong evidence for an obligatory exchange me chanism. We conclude that there is high-level expression of HAE1 funct ional activity in recombinant baculovirus-infected Sf-9 cells and that this system will prove useful for kinetic and structural analyses of the HAE1 protein. (C) 1996 Academic Press, Inc.