S4: Expression of antibodies in SHuffle

SHuffle is an engineered E. coli expression strain that can express complex, multi-disulfide bonded proteins in its cytoplasm. This is due to 1) the disruption of the two main reductive pathways in its cytoplasm 2) cytoplasmic over-expression of disulfide bond isomerase (DsbC) which corrects mis-oxidized disulfide bonds.

Antibodies require disulfide bonds for their correct folding. Therefore many types of antibody fragments including full-length IgG have been expressed only in the oxidative compartment of E. coli, the periplasm. But in order to achieve the maximal production, expression of a targeted protein in E. coli cytoplasm is desirable. Expression of the full-length antibodies in E. coli cytoplasm has thus far not been successful. Here we demonstrate the first successful expression of full length IgG in the cytoplasm of SHuffle strain and use the power of E. coli genetics to engineer novel chimeric constructs.