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Chemically competent E. coli cells suitable for T7 protein expression with enhanced capacity to correctly fold proteins with multiple disulfide bonds in the cytoplasm.
T7 expression
Engineered E. coli B strain to promote disulfide bond formation in the cytoplasm
Chemically competent E. coli B cells engineered to form proteins containing disulfide bonds in the cytoplasm. Suitable for T7 promoter driven protein expression.
Figure 1: vtPA activity assayed from crude lysates
Truncated tissue plasminogen activator (vtPA), which contains nine disulfide bonds when folded and oxidized correctly, was expressed from a pTrc99a plasmid in the cytoplasm of E. coli cells. After induction, cells were harvested and crude cell lysates were prepared. vtPA was assayed using a chromogenic substrate Chromozym t-PA (Roche #11093037001) and standardized to protein concentration using Bradford reagent. E. coli wt+ cells are DHB4, which is the parent of FÅ113 (Origami™).
Figure 2: PfCHT1 chitinase activity assayed from crude lysates
Plasmodium falciparum chitinase (PfCHT1) with three cysteines were expressed from a plasmid under the regulation of T7 promoter. After induction, cells were harvested and crude cell lysates were prepared. PfCHT1 was assayed using a chromogenic substrate (CalBioChem #474550) and standardized to protein concentration using Bradford reagent.
Highlights
Engineered E. coli B strain to promote disulfide bond formation in the cytoplasm
Constitutively expresses a chromosomal copy of the disufide bond isomerase DsbC
DsbC promotes the correction of mis-oxidized proteins into their correct form (1,3)
The cytoplasmic DsbC is also a chaperone that can assist in the folding of proteins that do not require disulfide bonds (4)
Enhanced BL21 derivative
Expresses a chromosomal copy of T7 RNAP
Activity of nonspecific endonuclease I (endA1) eliminated for highest quality plasmid preparations
Deficient in proteases Lon and OmpT
Resistance to phage T1 (fhuA2), Nit, Spec, Str*
*Resistance to low levels of streptomycin may be observed.
STORAGE AND HANDLING: Competent cells should be stored at -80°C. Storage at -20°C will result in a significant decrease in transformation efficiency. Cells lose efficiency whenever they are warmed above -80°C, even if they do not thaw.
References
Bessette, P.H. et al. (1999). Proc. Natl. Acad. Sci. USA. 96, 13703-13708.
Qiu, J., Swartz, J.R. and Georgiou, G. (1999). Appl. Environ. Microbiol. 64, 4891-4896.
Levy, R. et al. (2001). Protein Expr. Purif. 23, 338-347.
Chen, J. et al. (1999). J. Biol. Chem. 274, 19601-19605.
Boyd, D. et al. (2000). J. Bacteriol. 182, 842-847.
de Marco, A. (2009). Microbial Cell Factories. 8, 26.
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 J Biol Chem; 294(48), 18046-18056.. PubMedID: 31604819, DOI: 10.1074/jbc.RA119.011008
Agrawal, A., Bisharyan, Y., Papoyan, A, Bednenko, J., Cardarelli, J., Yao, M., Clark, T., Berkmen, 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 Expr Purif; 153, 7-17. PubMedID: 30081196, DOI: 10.1016/j.pep.2018.08.001.
Ren, G., Ke, N. and Berkmen, M. (2016) Use of the Shuffle Strains in Production of Proteins. Curr Protoc Protein Sci; Aug 1, 1;85:5.26.1-5.26.21.. PubMedID: 27479507 , DOI: 10.1002/cpps.11.
Anton, B.P., Fomenkov, A., Raleigh, E.A. and Berkmen, M. (2016) Complete Genome Sequence of the Engineered Escherichia coli SHuffle Strains and Their Wild-Type Parents Genome Announc; Mar 31;4(2), PubMedID: 27034504, DOI: 10.1128/genomeA.00230-16.
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), 992-1002. PubMedID: 30060704, DOI: 10.1080/19420862.2018.1496879
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 Nat Commun; (6)8072, PubMedID: 26311203, DOI: 10.1038/ncomms9072.
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Licenses
The buyer and user have a non-exclusive sub-license to use this system or any component thereof for RESEARCH PURPOSES ONLY, based upon agreement to the following assurances.
Transfer of the host cells that contain the cloned copy of the T7 gene 1 to third parties is explicitly prohibited. This limitation applies to E. coli ER2566, ER2833, ER3011, ER3012, ER3013 and ER3021, SHuffle T7, SHuffle T7 LysY, SHuffle T7 Express, SHuffle T7 Express LysY and their competent derivatives, C2566, C2833, C3010, C3013, C3016, C3022, C3026, C3027, C3029 and C3030 when provided separately or when provided in combination with appropriate vectors for said systems.
A license to use this system or any components thereof for commercial purposes may be obtained from New England Biolabs, Inc.
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