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Protein Science (2005), 14:2964-2971. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society
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Gateway vectors for the production of combinatorially-tagged His6-MBP fusion proteins in the cytoplasm and periplasm of Escherichia coli

Sreedevi Nallamsetty, Brian P. Austin1, Kerri J. Penrose1 and David S. Waugh

Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA

(RECEIVED July 21, 2005; FINAL REVISION September 28, 2005; ACCEPTED September 28, 2005)

Many proteins that accumulate in the form of insoluble aggregates when they are overproduced in Escherichia coli can be rendered soluble by fusing them to E. coli maltose binding protein (MBP), and this will often enable them to fold in to their biologically active conformations. Yet, although it is an excellent solubility enhancer, MBP is not a particularly good affinity tag for protein purification. To compensate for this shortcoming, we have engineered and successfully tested Gateway destination vectors for the production of dual His6MBP-tagged fusion proteins in the cytoplasm and periplasm of E. coli. The MBP moiety improves the yield and solubility of its fusion partners while the hexahistidine tag (His-tag) serves to facilitate their purification. The availability of a vector that targets His6MBP fusion proteins to the periplasm expands the utility of this dual tagging approach to include proteins that contain disulfide bonds or are toxic in the bacterial cytoplasm.

Keywords: immobilized metal affinity chromatography; IMAC; maltose-binding protein; Gateway cloning; fusion tag; structural genomics; TEV protease; expression vector; fusion protein

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051718605.

Reprint requests to: David S. Waugh, National Cancer Institute at Frederick, P.O. Box B, Frederick, MD 21702, USA; e-mail: waughd{at}ncifcrf.gov; fax: (301) 846-7148.


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