Improvement of soluble expression of anti-PD-1/CTLA-4 diabody in the cytoplasm of Escherichia coli.

Escherichia coli is one of the most efficient and widely used expression systems for the production of biopharmaceuticals. Despite all its many advantages, E. coli cannot efficiently produce complex, high molecular weight proteins produce complexmonoclonal antibodies and diabodies. In this study, the cytoplasmic soluble expression of a previously designed anti-PD-1/CTLA-4 single chain diabody (scDb) was evaluated at different incubation temperatures (15, 23, 30, and 37 °C) and IPTG concentrations (0.1, 0.5, and 1 mM) in E. coli BL21(DE3), Origami B(DE3), and SHuffle T7 strains. The effect of co-expression of the DnaK, DnaJ and GrpE chaperone on soluble protein expression was also investigated in E. coli BL21(DE3). The results showed that E. coli SHuffle T7 produced the highest expression of soluble protein. Lower incubation temperature increased the yield of soluble protein in all strains, whereas IPTG concentration had a negligible effect. Co-expression of chaperones in the E. coli BL21(DE3) also improved protein solubility compared with expression of the scDb alone. These findings suggest that E. coli SHuffle T7 is a promising host for the production of recombinant proteins production. In addition, chaperone co-expression and reduced incubation temperature can be considered effective strategies for higher soluble protein production.