TY - JOUR
T1 - Expression and characterization of the human erythrocyte anion exchanger in a baculovirus/Sf-9 cell system
AU - Dale, William E.
AU - Textor, Jacquelyn A.
AU - Mercer, Robert W.
AU - Simchowitz, Louis
N1 - Funding Information:
We acknowledge the expert technical assistance of Kristie Chis-cano and Sherri Vogt and the secretarial skills of Annette Irving. We are especially grateful to Dr. Paul De Weer for providing the computer programs and least-squares ®ts for data analysis. This work was supported by the Department of Veterans Affairs and by NIH Grant GM-39746.
PY - 1996/2
Y1 - 1996/2
N2 - 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 (K0.5 = 34 μM) 36Cl- uptake that was not present in uninfected cells. We also found that extracellular SO4/2- reduced 36Cl- influx [K0.5(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.
AB - 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 (K0.5 = 34 μM) 36Cl- uptake that was not present in uninfected cells. We also found that extracellular SO4/2- reduced 36Cl- influx [K0.5(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.
UR - http://www.scopus.com/inward/record.url?scp=0030087575&partnerID=8YFLogxK
U2 - 10.1006/prep.1996.0001
DO - 10.1006/prep.1996.0001
M3 - Article
C2 - 9172773
AN - SCOPUS:0030087575
VL - 7
SP - 1
EP - 11
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
IS - 1
ER -