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 ~0.5 x 10⁶ 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 [K(m)(Cl^-) = 44 mM; V(max)(Cl^-) = 48 mEq/liter of cell water·min] and H₂DIDS-inhibitable (K_0.5 = 34 μM) 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.