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T7 Expression

Within the realm of E. coli expression, the T7 system is the most popular approach for producing recombinant protein. In this system, the target gene is cloned into an expression vector downstream of the T7 promoter and this construct is introduced into a T7 expression host. T7 expression hosts such as DE3 prophage 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 target gene transcription. T7 expression is often very robust but may suffer from undesirable basal (non-induced) transcription especially in DE3 strains. Regulation of IPTG-induced T7 expression is accomplished by co-expressing the lac repressor from a plasmid or a host-encoded lacI gene and by co-expressing T7 lysozyme, the natural inhibitor of T7 RNA polymerase activity. T7 lysozyme may be expressed from pLysS or pLysE plasmids or a variant T7 lysozyme may be expressed from the lysY gene present within multiple NEB protein expression strains. The lysY gene product lacks amidase (lysozyme) activity against the E. coli cell wall while retaining the ability to inhibit basal T7 RNA polymerase activity. 

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    Publications related to T7 Expression
  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. 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.
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.
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