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Publisher
Humana Press, Totowa, NJ
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Authors: David M Rancour Steven K Backues Sebastian Y Bednarek
Publish Date: 2010
Volume: , Issue: , Pages: 3-20
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 costeffective 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 27954264–54274 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 coliexpressed 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 productionThis 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//wwwgreatlakesbioenergyorg which is supported by the US Department of Energy Office of Science Office of Biological and Environmental Research through Cooperative Agreement DEFC0207ER64494 between The Board of Regents of the University of Wisconsin System and the US Department of Energy
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