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A generic protocol for the expression and purification of recombinant proteins in Escherichia coli using a combinatorial His6-maltose binding protein fusion tag

Nature Protocols volume 2, pages 383–391 (2007)Cite this article

Abstract

We describe a generic protocol for the overproduction and purification of recombinant proteins in Escherichia coli. The strategy utilizes a dual His6-maltose binding protein (HisMBP) affinity tag that can be removed from the target protein by digestion of the fusion protein at a designed site by tobacco etch virus protease. The MBP moiety serves to enhance the solubility and promote the proper folding of its fusion partners, and the polyhistidine tag facilitates its purification to homogeneity. This protocol is divided into three stages, each of which takes approximately 1 week to complete: (i) construction of a HisMBP fusion vector; (ii) a pilot experiment to assess the yield and solubility of the target protein; and (iii) the large-scale production and purification of the target protein.

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Figure 1: Schematic illustration of the strategy involved in generating a PCR product for recombinational cloning.
Figure 2: Gateway cloning steps involved in the construction of a His6-maltose binding protein (HisMBP) fusion protein expression vector.
Figure 3: Intracellular processing of His6-maltose binding protein (HisMBP) fusion proteins by tobacco etch virus (TEV) protease.
Figure 4: Generic purification of Yersinia pestis LcrH, performed as described in Step 16B (protein purification).

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Acknowledgements

This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.

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  1. Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, P.O. Box B, Frederick, 21702-1201, Maryland, USA

    Sreedevi Nallamsetty & David S Waugh

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  1. Sreedevi Nallamsetty
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Correspondence to David S Waugh.

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Nallamsetty, S., Waugh, D. A generic protocol for the expression and purification of recombinant proteins in Escherichia coli using a combinatorial His6-maltose binding protein fusion tag. Nat Protoc 2, 383–391 (2007). https://doi.org/10.1038/nprot.2007.50

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