High-throughput protein expression screening and purification in Escherichia coli

Methods. 2011 Sep;55(1):65-72. doi: 10.1016/j.ymeth.2011.08.010. Epub 2011 Sep 8.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Ciona intestinalis / chemistry*
  • Cloning, Molecular
  • DNA-Binding Proteins / biosynthesis*
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / isolation & purification
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Gene Expression*
  • Genetic Vectors / chemistry
  • Genetic Vectors / genetics
  • Genetic Vectors / metabolism
  • High-Throughput Screening Assays*
  • Histidine / metabolism
  • Humans
  • Mice
  • Oligopeptides / metabolism
  • Protein Processing, Post-Translational
  • Protein Structure, Tertiary
  • Proteomics / methods*
  • Recombinant Fusion Proteins / biosynthesis*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Solubility
  • Thioredoxins / metabolism


  • DNA-Binding Proteins
  • His-His-His-His-His-His
  • Oligopeptides
  • Recombinant Fusion Proteins
  • Histidine
  • Thioredoxins