Strategies for protein coexpression in Escherichia coli

Tolia, N.H., Joshua-Tor, L. (January 2006) Strategies for protein coexpression in Escherichia coli. Nat Methods, 3 (1). pp. 55-64. ISSN 1548-7091 (Print)

DOI: 10.1038/nmeth0106-55


E. coli is a convenient host for heterologous protein expression. Its advantages include high levels of heterologous gene expression and scalability of experiments, low cost, fast growth, a lack of posttranslational modification and an ability to express labeled (isotope or seleno-methionine) proteins. However, heterologous gene expression in E. coli can lead to the production of insoluble and/or nonfunctional target proteins. This is often due to the absence of cofactors or post-translational modifications required for function, stability or folding. Coexpression of multiple genes in E. coli, such as the members of a stable multiprotein complex1 or a protein with a chaperone2,3, can in many cases alleviate these problems. Coexpression involves the transformation of E. coli with several plasmids that have compatible origins of replication and independent antibiotic selection for maintenance. The Duet (Novagen) vectors have two multiple cloning sites per vector, five compatible origins of replication and four antibiotic selection markers, allowing the simultaneous expression of up to eight proteins. The combination of Duet vectors with other commercial plasmids allows the use of affinity tags, such as glutathione S-transferase (GST) or maltose binding protein (MBP), which can ease the recovery and improve the solubility of the desired target. Coexpression in E. coli therefore provides a useful alternative to the complicated and expensive expression systems, such as yeast, baculovirus or mammalian cell culture, which are commonly used to overcome problems of heterologous protein expression. A summary of the method is presented in Figure 1.

Item Type: Paper
Uncontrolled Keywords: Animals Carrier Proteins chemistry genetics Cloning Molecular methods Escherichia coli chemistry genetics metabolism Genetic Vectors/genetics Glutathione Transferase chemistry genetics Humans Molecular Chaperones biosynthesis genetics Protein Folding Recombinant Proteins biosynthesis genetics isolation & purification
Subjects: organism description > bacteria > escherichia coli
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein expression
CSHL Authors:
Communities: CSHL labs > Joshua-Tor lab
School of Biological Sciences > Publications
Depositing User: CSHL Librarian
Date: January 2006
Date Deposited: 08 Dec 2011 18:31
Last Modified: 26 Sep 2014 14:32
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