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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 and with enhanced reduction of basal expression.
T7 expression
Engineered E. coli B strain to promote disulfide bond formation in the cytoplasm
Control of T7 RNA Polymerase by lysozyme allows potentially toxic genes to be expressed
Deficient in proteases Lon and OmpT
Resistant to phage T1 (fhuA2)
Free of animal products
No dry ice surcharge with NEB competent cell shipments
Chemically competent E. coli B cells engineered to form proteins containing disulfide bonds in the cytoplasm. Suitable for T7 promoter driven protein expression of toxic proteins.
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)
Expresses a chromosomal copy of T7 RNAP
Inactive mutant lysozyme expressed from miniF plasmid
Allows for the expression of toxic proteins
Enhanced BL21 derivative
Resistance to phage T1 (fhuA2), Cam, Nit, Spec, and Str*
*Resistance to low levels of streptomycin may be observed.
Enhanced capacity to correctly fold proteins with multiple disulfide bonds in the cytoplasm
Application Features
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 Brac3026dford reagent.
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.
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
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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.
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
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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