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 (His6–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 His6–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 His6–MBP tag are presented.
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Acknowledgments
This project was supported 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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Sun, P., Tropea, J.E., Waugh, D.S. (2011). Enhancing the Solubility of Recombinant Proteins in Escherichia coli by Using Hexahistidine-Tagged Maltose-Binding Protein as a Fusion Partner. In: Evans, Jr., T., Xu, MQ. (eds) Heterologous Gene Expression in E.coli. Methods in Molecular Biology, vol 705. Humana Press. https://doi.org/10.1007/978-1-61737-967-3_16
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DOI: https://doi.org/10.1007/978-1-61737-967-3_16
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