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.
SHuffle strains can correctly fold disulfide bonds in the cytoplasm
Disulfide bond formation in the cytoplasm of wild type E. coli is not favorable, while SHuffle is capable of correctly folding proteins with multiple disulfide bonds in the cytoplasm.
Figure 1: Express higher levels of biologically active protein with SHuffle.
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: SHuffle offers robustness and less toxicity than origami strains.
Plasmodium falciparum chitinase (PfCHT1) with three cysteines was 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.
*Ideally, DNA for transformation should be purified and resuspended in water or TE. However, up to 10 µl of DNA directly from a ligation mix can be used with only a two-fold loss of transformation efficiency. Where it is necessary to maximize the number of transformants (e.g. a library), a purification step, either a spin column or phenol/chloroform extraction and ethanol precipitation should be added.
Oxidizing cytoplasmic environment enables disulfide bond formation
DsbC (disulfide bond isomerase) directs correct disulfide bond formation
DsbC also acts as a chaperone for protein folding
Cytoplasmic expression increases protein yield of disulfide bonded proteins
A wide range of antibiotics can be used for plasmid maintenance (Amp
s, Kan s, Tet s, Cam s [except for lysY versions]) Transformation efficiency:
1 x 10
6 cfu/µg pUC19 DNA (SHuffle) 1 x 10
7 cfu/µg pUC19 DNA (SHuffle Express) Protease deficient
TI phage resistant (
fhuA2) Free of animal products
MiniF lacIq (CamR) / fhuA2 [lon] ompT gal sulA11 R(mcr-73::miniTn10--TetS)2 [dcm] R(zgb-210::Tn10--TetS) endA Δ(mcrC-mrr)114::IS10