title: Medium-throughput production of recombinant human proteins: protein production in E. coli.
abstract: In Chapter 4 we described the 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 crystallization 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.
authors.affiliation: Structural Genomics Consortium, University of Oxford, Oxford, UK.
authors.forename: Nicola A
authors.initials: NA
authors.lastname: Burgess-Brown
authors.forename: Pravin
authors.initials: P
authors.lastname: Mahajan
authors.forename: Claire
authors.initials: C
authors.lastname: Strain-Damerell
authors.forename: Opher
authors.initials: O
authors.lastname: Gileadi
authors.forename: Susanne
authors.initials: S
authors.lastname: Gräslund
mesh_terms: D061249:Batch Cell Culture Techniques; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D011786:Quality Control; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D014169:Transformation, Bacterial
chemical_list: D011506:Proteins; D011993:Recombinant Fusion Proteins