title: Purification of dual-tagged intact recombinant proteins.
abstract: Large-scale purification of recombinant proteins has been used extensively to assist numerous protein studies, including investigation of function, substrate identification and protein-protein interaction of low abundance proteins. Genetic fusion of affinity tags to these proteins has also been widely used for ease of purification by affinity chromatography. However, this technique sometimes yields unstable and degraded protein products limiting its application. In this study, we show a facile and straightforward method of dual-tagged recombinant protein purification that eliminates contamination by degraded protein products. A 6His-containing BamHI-HindIII fragment from pQE12 was ligated into the pGEX-KG BamHI-HindIII fragment and the protein of interest (p25(nck5a), which is highly susceptible to proteolytic degradation when expressed and purified from bacteria) was cloned into the BamHI site without a termination codon. The resulting plasmid construct, designated as pGST-p25(nck5a)-6His, with GST at the N-terminal and 6His at the C-terminal was expressed in Escherichia coli DH5alpha and purified using a two-step procedure. We show that using Ni(2+)-NTA chromatography as a first purification step and GSH-agarose chromatography as a second step, rather than vice-versa, yields a highly purified intact protein that is free of any contaminating degraded protein product. The purified fusion protein is soluble and fully active.
authors.affiliation: Department of Cell Biology and Anatomy, University of Calgary, Calgary, Alberta, Canada.
authors.forename: J L
authors.initials: JL
authors.lastname: Rosales
authors.forename: K Y
authors.initials: KY
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase