Abstract
Escherichia coli is a frequently used expression system for the generation of protein encoded by genes from diverse kingdoms and, thus, it is well suited for the production of protein antigens for antibody generation. It is a system of choice for many due to factors such as (1) the commercial availability of a vast array of reagents and materials needed for cloning, expression, and purification and (2) the potential high protein yields that can be acquired in a timely and cost-effective manner. This chapter will focus on (1) the general principles to keep in mind when choosing an antigen to express and (2) the use of a modified pGEX vector system (Rancour et al., J. Biol. Chem. 279:54264–54274, 2004) to use in its expression. Simplified protocols are provided for (1) assessing the expression of your protein, (2) testing whether your protein is or is not expressed as a soluble product, (3) performing bulk purifications of soluble or insoluble E. coli-expressed protein to acquire enough to be used for a complete immunization protocol, and (4) an optional procedure for epitope tag removal from your expressed protein of interest in order to avoid the unnecessary and sometimes unwanted production of antibodies against the fusion protein affinity chromatography tag. These four procedures have been used extensively and successfully in our lab as a basis for the production of recombinant protein and subsequent antibody production.
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Acknowledgments
This work was supported in part by the Department of Energy, Division of Energy Biosciences (Grant no. DE–FG02–ER20332 to S. Y. B.), and the DOE Great Lakes Bioenergy Research Center (http://www.greatlakesbioenergy.org), which is supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through Cooperative Agreement DE-FC02-07ER64494 between The Board of Regents of the University of Wisconsin System and the US Department of Energy.
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Rancour, D.M., Backues, S.K., Bednarek, S.Y. (2010). Protein Antigen Expression in Escherichia coli for Antibody Production. In: Schwartzbach, S., Osafune, T. (eds) Immunoelectron Microscopy. Methods in Molecular Biology, vol 657. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-783-9_1
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DOI: https://doi.org/10.1007/978-1-60761-783-9_1
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