Abstract
In Chapter 3, we described the Structural Genomics Consortium (SGC) process for generating multiple constructs of truncated versions of each protein using LIC. In this chapter we provide a step-by-step procedure of our E. coli system for test expressing intracellular (soluble) proteins in a 96-well format that enables us to identify which proteins or truncated versions are expressed in a soluble and stable form suitable for structural studies. In addition, we detail the process for scaling up cultures for large-scale protein purification. This level of production is required to obtain sufficient quantities (i.e., milligram amounts) of protein for further characterization and/or structural studies (e.g., crystallization or cryo-EM experiments). Our standard process is purification by immobilized metal affinity chromatography (IMAC) using nickel resin followed by size exclusion chromatography (SEC), with additional procedures arising from the complexity of the protein itself.
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Acknowledgments
We would like to thank all the SGC scientists (past and present) who contributed toward the development of the method. The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative Medicines Initiative (EU/EFPIA), Janssen, Merck KGaA, MSD, Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and Wellcome.
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Burgess-Brown, N.A., Mahajan, P., Strain-Damerell, C., Fernandez-Cid, A., Gileadi, O., Gräslund, S. (2021). Screening and Production of Recombinant Human Proteins: Protein Production in E. coli. In: Chen, Y.W., Yiu, CP.B. (eds) Structural Genomics. Methods in Molecular Biology, vol 2199. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0892-0_4
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DOI: https://doi.org/10.1007/978-1-0716-0892-0_4
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