title: High-throughput protein expression screening and purification in Escherichia coli.
abstract: Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, to obtain milligrams of soluble proteins is still challenging since many proteins are expressed in an insoluble form without optimization. Therefore when working with tens of proteins or protein domains it is recommended that high-throughput expression screening at a small scale (1-4ml of culture) is carried out to identify the optimal conditions for soluble protein production. Once determined, these culture conditions can be applied at a large scale to produce sufficient protein for structural or functional studies. We describe a procedure that has enabled the systematic screening of culture conditions or fusion-tags on hundreds of cultures per week. The analysis of the optimal conditions for the soluble production of these proteins helped us to design a simple and efficient protocol for soluble protein expression screening. This protocol has since been used on hundreds of proteins and is illustrated with the genome wide scale production of proteins containing the DNA binding domains of Ciona intestinalis.
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, UMR6098 CNRS, Université de Provence and Université de la Méditerranée, Case 932, 163 Avenue de Luminy, 13288 Marseille cedex 9, France. renaud.vincentelli@afmb.univ-mrs.fr
authors.forename: Renaud
authors.initials: R
authors.lastname: Vincentelli
authors.forename: Agnès
authors.initials: A
authors.lastname: Cimino
authors.forename: Arie
authors.initials: A
authors.lastname: Geerlof
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kubo
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Satou
authors.forename: Christian
authors.initials: C
authors.lastname: Cambillau
mesh_terms: D000818:Animals; D002938:Ciona intestinalis / Q000737:chemistry*; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006639:Histidine / Q000378:metabolism; D006801:Humans; D051379:Mice; D009842:Oligopeptides / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D013879:Thioredoxins / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D013879:Thioredoxins