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Protein Expression in E. Coli

Protein expression in the bacterium E. coli is the most popular means of producing recombinant protein. E. coli is a well-established host that offers short culturing time, easy genetic manipulation and low cost media. Additionally, E. coli has a long history of being capable of producing a wide variety of different types of proteins. Even proteins that contain disulfide bonds can be expressed within the cytoplasm of NEB SHuffle® strains.

Within the realm of E. coli expression, the T7 system is the most popular approach for producing proteins. In this system, an expression vector containing a gene of interest cloned downstream of the T7 promoter is introduced into a T7 expression host. T7 expression hosts such as DE3 strains or T7 Express strains carry a chromosomal copy of the phage T7 RNA polymerase gene. When inducer is added, T7 RNA polymerase is expressed and becomes dedicated to transcription of the gene of interest.
 

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Protein Expression in E. Coli includes these areas of focus:
T7 Expression
Non-T7 Expression
FAQs for Protein Expression in E. Coli
Protocols for Protein Expression in E. Coli
    Publications related to Protein Expression in E. Coli
  1. Agrawal, A., Bisharyan, Y., Papoyan, A, Bednenko, J., Cardarelli, J., Yao, M., Clark, T., Berkm​en, M., Ke, N., Colussi, P. 2019. Fusion to Tetrahymena thermophila granule lattice protein 1 confers solubility to sexual stage malaria antigens in Escherichia coli. Protein Expression and Purification. 153, PubMedID: 30081196, DOI: 10.1016/j.pep.2018.08.001.
  2. Leith, E.M., O'Dell, W.B., Ke, N., McClung, C., Berkmen, M., Bergonzo, C., Brinson, R.G., Kelman, Z 2019. Characterization of the internal translation initiation region in monoclonal antibodies expressed in Escherichia coli Journal of Biological Chemistry. 294(48), PubMedID: 31604819, DOI: 10.1074/jbc.RA119.011008
  3. Reddy, P.T., Brinson, R.G., Hoopes, J.T., McClung, C., Ke, N., Kashi, L. 2018. Platform development for expression and purification of stable isotope labeled monoclonal antibodies in Escherichia coli. mAbs Mabs. 10 (7), PubMedID: 30060704, DOI: 10.1080/19420862.2018.1496879
  4. Ke, Na; Berkmen, Mehmet; Ren, Guoping; 2017. A water-soluble DsbB variant that catalyzes disulfide-bond formation in vivo Nature Chemical Biology. 13, PubMedID: 28628094, DOI: 10.1038/nchembio.2409
  5. Ren, G., Ke, N. and Berkmen, M. 2016. Use of the Shuffle Strains in Production of Proteins. Curr Protoc Protein Sci.. Aug 1, PubMedID: 27479507 , DOI: 10.1002/cpps.11.
  6. Robinson, M.-P., Ke, N., Lobstein, J., Peterson, C., Szkodny, A., Mansell, T.J., Tuckey, C., Riggs, P.D., Colussi, P.A., Noren, C.J., Taron, C.H., Delisa, M.P., Berkmen, M. 2015. Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria Nature Communications . (6)8072, PubMedID: , DOI: 10.1038/ncomms9072.
  7. Hemmis, C.W., Berkmen, M., Eser, M.and Schildbach, J.F. 2011. TrbB from conjugative plasmid F is a structurally distinct disulfide isomerase that requires DsbD for redox state maintenance. J Bacteriol. 193(18), PubMedID: 21742866, DOI: 10.1128/JB.00351-11
  8. Shouldice, S.R., Cho, S.H., Boyd, D., Heras, B., Eser, M., Beckwith, J., Riggs, P., Martin, J.L.and Berkmen, M. 2010. In vivo oxidative protein folding can be facilitated by oxidation-reduction cycling. Mol.Microbiol.. 75(1), PubMedID: 19968787, DOI:
  9. Manta, Bruno; Berkmen, Mehmet; . Disulfide Bond Formation in the Periplasm of Escherichia coli EcoSal Plus. , PubMedID: , DOI: 10.1128/ecosalplus.ESP-0012-2018.
NiCo21(DE3) outperforms BL21(DE3) for His-tagged protein purity
 
His-tagged Glutamyl-tRNA synthetase (GluRS) was expressed in BL21(DE3) and NiCo21(DE3) under identical conditions, followed by target protein isolation by Ni-NTA resin (Qiagen). Ni-NTA elution fractions (E) have different E. coli contaminants because three essential contaminant proteins are tagged with chitin binding domain (CBD) within NiCo21(DE3). The NiCo21(DE3) elution was incubated with chitin resin, improving GluRS purity to 97% (FTc). Protein purity was assessed using a LabChip GXII system (Perkin Elmer). M= ColorPlus prestained protein ladder; L = Ni-NTA Load; ft = Ni-NTA flow-through; E = Ni-NTA elution, FTc = chitin column flow-through. Reference: Robichon et al. (2011) Appl. Environ. Microbiol. 77:4634-4646.
T7 Controlled Expression of Protein in
E. coli Hosts
A T7 expression plasmid containing a gene encoding E. coli UDG was transformed into each host, grown to 0.6 OD and induced for 3 hours. Comparison of soluble extracts from uninduced (-) and induced (+) cells shows superior control of expression in the T7 Express hosts while maintaining high levels of induced expression.
The Lemo System™ for Membrane Protein Expression
Lemo System™ enables simple, rapid optimization of membrane protein expression.
Disulfide Bond Formation

Disulfide bond formation in the cytoplasm of wild type E. coli is not favorable, while SHuffle is capable of correctly folding proteins with multiple disulfide bonds in the cytoplasm.

NiCo21(DE3) Two-Step Purification
Lemo21(DE3) achieves a higher level of expression than BL21(DE3) pLysS.
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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.


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