Abstract

In the field of biotechnology, fusing recombinant proteins to highly soluble partners is a common practice for overcoming aggregation in Escherichia coli. E. coli maltose-binding protein (MBP) has been recognized as one of the most effective solubilizing agents, having frequently been observed to improve the yield, enhance the solubility, and promote the proper folding of its fusion partners. The use of a dual hexahistidine-maltose-binding protein affinity tag (His(6)-MBP) has the additional advantage of allowing the fusion protein to be purified by immobilized metal affinity chromatography (IMAC) instead of or in addition to amylose affinity chromatography. This chapter describes a generic method for the overproduction of combinatorially tagged His(6)-MBP fusion proteins in E. coli, with particular emphasis on the use of recombinational cloning to construct expression vectors. In addition, simple methods for evaluating the solubility of the fusion protein and the passenger protein after it is cleaved from the dual His(6)-MBP tag are presented.

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