Abstract
Insolubility of recombinant proteins in Escherichia coli is a major impediment to their production for structural and functional studies. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely recognized as a premier solubilizing agent. In this chapter, we describe how to construct dual His6-MBP-tagged fusion proteins by Gateway® recombinational cloning and how to predict their yield and solubility. We also describe a simple and rapid procedure to test the ability of a His6-MBP fusion protein to bind to Ni-NTA resin and to be digested by tobacco etch virus (TEV) protease, along with a method to assess the solubility of the target protein after it has been separated from His6-MBP.
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Acknowledgements
We thank Di Zhang for constructing the His6-MBP-DHFR expression vector. This research was funded by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the US Government.
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Needle, D., Waugh, D.S. (2014). Rescuing Aggregation-Prone Proteins in Escherichia coli with a Dual His6-MBP Tag. In: Giannone, R., Dykstra, A. (eds) Protein Affinity Tags. Methods in Molecular Biology, vol 1177. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1034-2_7
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DOI: https://doi.org/10.1007/978-1-4939-1034-2_7
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-1034-2
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