DOUBLE TAGGING RECOMBINANT A(1)-ADENOSINE AND A(2A)-ADENOSINE RECEPTORS WITH HEXAHISTIDINE AND THE FLAG EPITOPE - DEVELOPMENT OF AN EFFICIENT GENERIC PROTEIN-PURIFICATION PROCEDURE

An expression plasmid for mammalian cells (CLDN10B) has been modified to add nucleotides encoding hexahistidine and the FLAG peptide (H/F) t o cDNAs. The new mammalian expression plasmid has been named pDouble T rouble (pDT). The plasmid and a recombinant baculovirus were used to p roduce native- and H/F- human A(1) and A(2A) adenosine receptors, opti mally expressed in CHO-K1 and Sf9 cells, respectively. Binding to reco mbinant H/F-A(1) receptors (B-max = 30 pmol/mg protein) was characteri zed using [H-3]8-cyclopentyl-1,3-dipropylxanthine ([H-3]CPX) and I-125 -N-6-aminobenzyladenosine (I-125-ABA). Binding to H/F-A(2A) receptors (B-max = 48 pmol/mg protein) was characterized using [H-3]5,-N-ethylca rboxamidoadenosine ([H-3]NECA) and [H-3]2-[4-(2- carboxyethyl)phenethy lamino]-NECA ([H-3]CGS21680). By comparison to native receptors, the a ddition of H/F to the amino termini of these receptors had no effect o n the binding affinities of radioligands or competing compounds. The f unction of A(1) adenosine receptors to reduce forskolin stimulated cyc lic AMP accumulation in intact cells was not affected by the H/F exten sion. Anti-FLAG and Ni-nitrilotriacetic acid affinity chromatography r esulted in high yield (>50% overall recovery) of nearly homogeneous (> 90% pure) receptors visible on silver stained gels that comigrated wit h photolabeled receptors before and after deglycosylation with N-glyco sidase F. We anticipate that pDT will be generally useful for facilita ting the purification in high yield of recombinant receptors and other proteins by single or sequential affinity chromatography steps.