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Screening and production of recombinant human proteins: protein production in E. coli

DOI: 10.1007/978-1-0716-0892-0_4 DOI Help

Authors: Nicola Burgess-brown (Structural Genomics Consortium, University of Oxford) , Pravin Mahajan (Astex Pharmaceuticals) , Claire Strain-damerell (Diamond Light Source) , Alejandra Fernandez-cid (Structural Genomics Consortium, University of Oxford) , Opher Gileadi (Structural Genomics Consortium, University of Oxford) , Susanne Gräslund (Structural Genomics Consortium, Karolinska Institutet)
Co-authored by industrial partner: Yes

Type: Book Chapter

Book Chapter: 4

State: Published (Approved)
Published: October 2020

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.

Journal Keywords: E. coli; Bacteria; Expression; Recombinant; Protein; Purification; Immobilized metal affinity chromatography (IMAC); Size exclusion chromatography (SEC); Gel filtration

Subject Areas: Biology and Bio-materials

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Added On: 03/11/2020 09:39

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