title: A rapid and universal tandem-purification strategy for recombinant proteins.
abstract: A major goal in the production of therapeutic proteins, subunit vaccines, as well as recombinant proteins needed for structure determination and structural proteomics is their recovery in a pure and functional state using the simplest purification procedures. Here, we report the design and use of a novel tandem (His)(6)-calmodulin (HiCaM) fusion tag that combines two distinct purification strategies, namely, immobilized metal affinity (IMAC) and hydrophobic interaction chromatography (HIC), in a simple two-step procedure. Two model constructs were generated by fusing the HiCaM purification tag to the N terminus of either the enhanced green fluorescent protein (eGFP) or the human tumor suppressor protein p53. These fusion constructs were abundantly expressed in Escherichia coli and rapidly purified from cleared lysates by tandem IMAC/HIC to near homogeneity under native conditions. Cleavage at a thrombin recognition site between the HiCaM-tag and the constructs readily produced untagged, functional versions of eGFP and human p53 that were >97% pure. The HiCaM purification strategy is rapid, makes use of widely available, high-capacity, and inexpensive matrices, and therefore represents an excellent approach for large-scale purification of recombinant proteins as well as small-scale protein array designs.
authors.affiliation: Department of Pharmaceutical Sciences, University of Toronto, Ontario M5S 3M2, Canada.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: McCluskey
authors.forename: Gregory M K
authors.initials: GM
authors.lastname: Poon
authors.forename: Jean
authors.initials: J
authors.lastname: Gariépy
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002147:Calmodulin / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000302:isolation & purification*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D016159:Tumor Suppressor Protein p53 / Q000302:isolation & purification*
chemical_list: D002147:Calmodulin; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins