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Published online before print October 26, 2007, 10.1110/ps.072894407
Protein Science (2007), 16:2726-2732. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society
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A rapid and universal tandem-purification strategy for recombinant proteins

Andrew J. McCluskey1,2,4, Gregory M.K. Poon2,4, and Jean Gariépy1,2,3

1 Department of Pharmaceutical Sciences, University of Toronto, Ontario M5S 3M2, Canada
2 Ontario Cancer Institute, University Health Network, Ontario M5G 2M9, Canada
3 Department of Medical Biophysics, University of Toronto, Ontario M5G 2M9, Canada

(RECEIVED March 25, 2007; FINAL REVISION September 5, 2007; ACCEPTED September 16, 2007)

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.

Keywords: protein purification; immobilized metal-affinity chromatography; hydrophobic interaction chromatography; tandem-affinity purification


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