Abstract
Protein expression and purification have traditionally been time-consuming, case-specific endeavors, and are considered to be the greatest bottlenecks in most proteomics pipelines. Escherichia coli (E. coli) is the most convenient and cost-effective host, although optimal conditions for the expression of different proteins vary widely. Proteins vary in their structural stability, solubility, and toxicity in this environment, resulting in differing rates of protein degradation, formation into insoluble inclusion bodies, and cell death, thus affecting the amount of soluble protein that can be obtained from E. coli grown in culture. To take full advantage of a variety of strategies developed to improve the expression of soluble protein in E. coli, an easy, rapid means to test many growth parameters is necessary. This chapter describes a dot-blot expression screen to test the effects of growth and induction parameters on the yield of soluble protein. The expression screen is used to detect hexahistidine-tagged proteins expressed in E. coli; however, it is adaptable for the detection of other affinity tags or fusion partners that have suitable antibodies available. In this example, induction time and temperature are tested; however, it can be used to test additional parameters, such as affinity tag type and placement, E. coli host type, and growth medium formulations.
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Acknowledgments
The author thanks Jennifer Massi and Shirin Fuller for technical assistance, and Michael Murphy, Peter Beernink, and Paul Richardson for reading of the manuscript. This work was performed under the auspices of the US Department of Energy, Office of Biological and Environmental Research, by the University of California, Lawrence Livermore National Laboratory (contract No. W-7405-Eng-48), Lawrence Berkeley National Laboratory (contract No. DEAC03-76SF00098), and Los Alamos National Laboratory (contract No. W-7405-ENG-36).
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Doyle, S.A. (2005). Screening for the Expression of Soluble Recombinant Protein in Escherichia coli . In: Zanders, E.D. (eds) Chemical Genomics. Methods in Molecular Biology™, vol 310. Humana Press. https://doi.org/10.1007/978-1-59259-948-6_8
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DOI: https://doi.org/10.1007/978-1-59259-948-6_8
Publisher Name: Humana Press
Print ISBN: 978-1-58829-399-2
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