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Chemically competent E. coli cells suitable for non-T7 protein expression with enhanced capacity to correctly fold proteins with multiple disulfide bonds in the cytoplasm.
Non-T7 expression
Engineered E. coli B strain to promote disulfide bond formation in the cytoplasm
The following reagents are supplied with this product:
NEB #
Component Name
Component #
Stored at (°C)
Amount
Concentration
C3028J
-80
SHuffle® Express Competent E. coli
C3028JVIAL
-80
12 x 0.05 ml
Not Applicable
Properties & Usage
Antibiotic for Plasmid Selection
Antibiotics for Plasmid Selection
Working Concentration
Ampicillin
100 µg/ml
Carbenicillin
100 µg/ml
Chloramphenicol
33 µg/ml
Kanamycin
30 µg/ml
Tetracycline
15 µg/ml
Shipping Notes
Ships on dry ice
Antibiotic Resistance
str (resistance to low levels of streptomycin may be observed)
nit
spec
Advantages and Features
Features
Protease deficient/B strain
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 Bradford 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.
References
Bessette, P.H. et al. (1999). Proc. Natl. Acad. Sci. USA. 96, 13703-13708.
Qiu, J., Swartz, J.R. and Georgiou, G. (1998). Appl. Environ. Microbiol. 64, 4891-4896.
Levy, R. et al. (2001). Protein Expr. Purif. 20, 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.
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.
Specifications & Change Notifications
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.
