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Updated: Jul 17, 2026
Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag
Published on: January 16, 2012
A generic method for the production of recombinant proteins in Escherichia coli using a dual
Joseph E Tropea1, Scott Cherry, Sreedevi Nallamsetty
1Protein Engineering Section, Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute--Frederick, MD, USA.
Abstract:
A generic protocol that utilizes a dual hexahistidine-maltose-binding protein (His6-MBP) affinity tag has been developed for the production of recombinant proteins in Escherichia coli. The MBP moiety improves the yield and enhances the solubility of the passenger protein while the His-tag facilitates its purification. The fusion protein (His6-MBP-passenger) is purified by immobilized metal affinity chromatography (IMAC) on nickel-nitrilotriacetic acid (Ni-NTA) resin and then cleaved in vitro with His6-tobacco etch virus protease to separate the His6-MBP from the passenger protein. In the final step, the unwanted byproducts of the digest are absorbed by a second round of IMAC, leaving nothing but the pure passenger protein in the flow-through fraction. Endogenous proteins that bind to the Ni-NTA resin during the first IMAC step also do so during the second round of IMAC. Hence, the application of two successive IMAC steps, rather than just one, is the key to obtaining crystallization-grade protein with a single affinity technique.
