PURIFICATION OF RECOMBINANT ANTIGENIC EPITOPES OF THE HUMAN 68-KDA (U1) RIBONUCLEOPROTEIN ANTIGEN USING THE EXPRESSION SYSTEM PH6EX3 FOLLOWED BY METAL CHELATING AFFINITY-CHROMATOGRAPHY

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 μg recombinant protein/ml of bacterial culture was obtained. © 1992 Academic Press, Inc.