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 solubiliz-ing 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.
CITATION STYLE
Needle, D., & Waugh, D. S. (2014). Rescuing aggregation-prone proteins in Escherichia coli with a dual His6-MBP tag. Methods in Molecular Biology, 1177, 81–94. https://doi.org/10.1007/978-1-4939-1034-2_7
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