title: Double tagging recombinant A1- and A2A-adenosine receptors with hexahistidine and the FLAG epitope. Development of an efficient generic protein purification procedure.
abstract: 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 pDoubleTrouble (pDT). The plasmid and a recombinant baculovirus were used to produce native-and H/F-human A1 and A2A adenosine receptors, optimally expressed in CHO-K1 and Sf9 cells, respectively. Binding to recombinant H/F-A1 receptors (Bmax = 30 pmol/mg protein) was characterized using [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]CPX) and 125I-N6-aminobenzyladenosine (125I-ABA). Binding to H/F-A2A receptors (Bmax = 48 pmol/mg protein) was characterized using [3H]5'-N-ethylcarboxamidoadenosine ([3H]NECA) and [3H]2-[4-(2-carboxyethyl)phenethylamino]-NECA ([3H]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 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 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.
authors.affiliation: Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
authors.forename: A S
authors.initials: AS
authors.lastname: Robeva
authors.forename: R
authors.initials: R
authors.lastname: Woodard
authors.forename: D R
authors.initials: DR
authors.lastname: Luthin
authors.forename: H E
authors.initials: HE
authors.lastname: Taylor
authors.forename: J
authors.initials: J
authors.lastname: Linden
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D019830:Adenosine-5'-(N-ethylcarboxamide); D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002846:Chromatography, Affinity / Q000379:methods; D006224:Cricetinae; D018076:DNA, Complementary; D000939:Epitopes / Q000032:analysis; D006639:Histidine; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008322:Mammals; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D010452:Peptide Biosynthesis; D010455:Peptides / Q000032:analysis; D010627:Phenethylamines / Q000378:metabolism; D010957:Plasmids; D011869:Radioligand Assay; D018047:Receptors, Purinergic P1 / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D016324:Sequence Tagged Sites; D018411:Spodoptera; D014162:Transfection; D014316:Tritium
chemical_list: D018076:DNA, Complementary; D000939:Epitopes; D007457:Iodine Radioisotopes; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D010455:Peptides; D010627:Phenethylamines; D018047:Receptors, Purinergic P1; D011994:Recombinant Proteins; D014316:Tritium; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; D019830:Adenosine-5'-(N-ethylcarboxamide); D006639:Histidine; C085706:FLAG peptide; D000241:Adenosine