Double tagging recombinant A(1)- 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) to cDNAs. The new mammalian expression plasmid has been named pDouble Trouble (pDT). The plasmid and a recombinant baculovirus were used to produce native- and H/F- human A(1) and A(2A) adenosine receptors, optimally expressed in CHO-K1 and Sf9 cells, respectively. Binding to recombinant H/F-A(1) receptors (B-max = 30 pmol/mg protein) was characterized 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-ethylcarboxamidoadenosine ([H-3]NECA) and [H-3]2-[4-(2- carboxyethyl)phenethylamino]-NECA ([H-3]CGS21680). By comparison to native receptors, the addition of H/F to the amino termini of these receptors had no effect on the binding affinities of radioligands or competing compounds. The function of A(1) adenosine receptors to reduce forskolin stimulated cyclic AMP accumulation in intact cells was not affected by the H/F extension. Anti-FLAG and Ni-nitrilotriacetic acid affinity chromatography resulted in high yield (>50% overall recovery) of nearly homogeneous (>90% pure) receptors visible on silver stained gels that comigrated with photolabeled receptors before and after deglycosylation with N-glycosidase F. We anticipate that pDT will be generally useful for facilitating the purification in high yield of recombinant receptors and other proteins by single or sequential affinity chromatography steps.