Abstract
Inducible production of proteins from cloned genes in E. coli is widely used, economical, and effective. However, common practices can result in unintended induction, inadvertently generating cultures that give poor or variable yields in protein production. Recipes are provided for (1) defined culture media in which expression strains grow to saturation without induction, thereby ensuring stable frozen stocks and seed cultures with high fractions of fully inducible cells, and (2) defined or complex media that maintain the same high fraction of inducible cells until auto-induction in late log phase to produce fully induced high-density cultures at saturation. Simply inoculating a suitable auto-inducing medium from such a seed culture and growing to saturation generally produces much higher levels of target protein per volume of culture than monitoring culture growth and adding IPTG or other inducer at the appropriate cell density. Many strains may be conveniently screened in parallel, and burdensome inoculation with fresh colonies, sometimes employed in hopes of assuring high yields, is entirely unnecessary. These media were developed for the T7 expression system using pET vectors in BL21(DE3) but are suitable or adaptable for other inducible expression systems in E. coli and for labeling proteins with selenomethionine for X-ray crystallography or with stable isotopes for NMR.
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References
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Acknowledgements
Work was supported by the Office of Biological and Environmental Research of the US Department of Energy, the Protein Structure Initiative of the National Institute of General Medical Sciences of the National Institutes of Health, as part of the New York Structural Genomics Research Consortium, and by internal research funding from Brookhaven National Laboratory.
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Studier, F.W. (2014). Stable Expression Clones and Auto-Induction for Protein Production in E. coli . In: Chen, Y. (eds) Structural Genomics. Methods in Molecular Biology, vol 1091. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-691-7_2
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DOI: https://doi.org/10.1007/978-1-62703-691-7_2
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Publisher Name: Humana Press, Totowa, NJ
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