1 A Generic Method for the Production of Recombinant Proteins in Escherichia coli Using a Dual Hexahistidine-Maltose-Binding Protein Affinity Tag

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 frac-tion. 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. Key Words: Maltose-binding protein; MBP; immobilized metal affinity chromatography; IMAC; His-tag; affinity tag; affinity chromatography; structural genomics; high throughput; tobacco etch virus protease; TEV protease. 1