title: Purification of recombinant antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein antigen using the expression system pH6EX3 followed by metal chelating affinity chromatography.
abstract: A novel plasmid expression vector (pH6EX3) that directs the synthesis of a fusion protein with a histidine hexapeptide at its N-terminus and a foreign protein at its C-terminus was constructed. The fusion gene is controlled by a strong tac promoter, leading to high-level expression of recombinant protein in several bacterial strains; the protein is deposited mainly as an insoluble mass in inclusion bodies. The fusion protein can be purified from the insoluble cell fraction by one-step affinity chromatography based on the selective interaction between the histidine hexapeptide and a metal chelating matrix charged with Ni2+ ions. The principle of this new system was tested by expressing and purifying antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein autoantigen. With the use of column chromatography and pH gradient elution, about 25 micrograms recombinant protein/ml of bacterial culture was obtained.
authors.affiliation: Department of Molecular Biology, ELIAS Entwicklungslabor, Freiburg, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Berthold
authors.forename: M
authors.initials: M
authors.lastname: Scanarini
authors.forename: C C
authors.initials: CC
authors.lastname: Abney
authors.forename: B
authors.initials: B
authors.lastname: Frorath
authors.forename: W
authors.initials: W
authors.lastname: Northemann
mesh_terms: D001483:Base Sequence; D002614:Chelating Agents; D002846:Chromatography, Affinity / Q000379:methods*; D000939:Epitopes / Q000302:isolation & purification*; D005822:Genetic Vectors; D006639:Histidine; D008969:Molecular Sequence Data; D009532:Nickel; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification*; D017411:Ribonucleoproteins, Small Nuclear / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D002614:Chelating Agents; D000939:Epitopes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D017411:Ribonucleoproteins, Small Nuclear; D006639:Histidine; D009532:Nickel