 Overview Advantages Expression Levels Characteristics
SHuffle® strains from NEB are engineered E. coli strains capable of expressing proteins with increasing disulfide bond complexity in the cytoplasm. SHuffle strains express the disulfide bond isomerase DsbC within the cytoplasm. DsbC isomerizes mis-oxidized substrates into their correctly folded state greatly enhancing the fidelity of disulfide bond formation. Cytoplasmic expression also results in significantly higher protein yields of disulfide bonded proteins when compared to periplasmic expression. SHuffle strains are sensitive to kan, amp, tet and in most cases, cam, which makes them able to express proteins from a wide variety of expression vectors offering greater versatility in experimental design.
NEB Recommends:
- SHuffle Express Strains
- 30° or lower
- Late log induction (OD600~0.8)
- For non-T7 promoters (e.g. lac and ara promoter expression vectors) we recommend the E.coli B strain, SHuffle Express (NEB #C3028H). For T7 promoter expression vectors, we recommend SHuffle T7 Express (NEB #C3029H). For toxic T7 promoter expression, we recommend SHuffle T7 Express lysY (NEB #C3030H). If SHuffle Express strains are not optimal, the E.coli K12 Shuffle strains are also available.
Advantages:
- Oxidizing cytoplasmic environment enables disulfide bond formation
- DsbC (disulfide bond isomerase) directs correct disulfide bond formation
- DsbC also acts as a general chaperone for protein folding
- Cytoplasmic expression increases protein yield
- A wide range of antibiotics can be used for plasmid maintenance (Amp, Kan, Tet, Cam [except for lysY versions])
- Transformation efficiency: 1 x 106 cfu/µg pUC19 DNA
- Protease deficient
- TI phage resistant (fhuA2)
- Free of animal products
Express higher levels of biologically active protein with SHuffle.
(A) 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™).
(B) Plasmodium falciparum chitinase (PfCHT1) with three cysteines.
* Resistance to low levels of streptomycin may be observed.
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