Strategies to optimize protein expression in E. coli

Abstract

Recombinant protein expression in Escherichia coli (E. coli) is simple, fast, inexpensive, and robust, with the expressed protein comprising up to 50 percent of the total cellular protein. However, it also has disadvantages. For example, the rapidity of bacterial protein expression often results in unfolded/misfolded proteins, especially for heterologous proteins that require longer times and/or molecular chaperones to fold correctly. In addition, the highly reductive environment of the bacterial cytosol and the inability of E. coli to perform several eukaryotic post-translational modifications results in the insoluble expression of proteins that require these modifications for folding and activity. Fortunately, multiple, novel reagents and techniques have been developed that allow for the efficient, soluble production of a diverse range of heterologous proteins in E. coli. This overview describes variables at each stage of a protein expression experiment that can influence solubility and offers a summary of strategies used to optimize soluble expression in E. coli.

Figure 1

Schematic overview of the topics covered in this review, highlighting the multiple parameters (listed on the right) that can greatly impact the success of soluble expression.

Figure 2

Flowchart of a general expression protocol used by the authors to express a broad range of targets, from phosphatases, to neuronal scaffolding proteins, to bacterial signaling proteins. The approximate time required to complete each segment of the protocol is listed to the left of the corresponding step.

Figure 3

Flowchart depicting the critical factors to consider, common obstacles, and potential solutions for each stage of protein expression in E. coli . The left column lists the major steps of recombinant protein expression with key variables to consider. The middle column includes common obstacles encountered at each step, while possible solutions for each obstacle are presented in the right column.