Abstract
Cost-effective production of therapeutic proteins in microbial hosts is an indispensable tool towards accessible healthcare. Many of these heterologously expressed proteins, including all antibody formats, require disulfide bond formation to attain their native and functional state. A system for catalyzed disulfide bond formation (CyDisCo) has been developed allowing efficient production of recombinant proteins in the cytoplasm of one of the most used microbial expression systems, Escherichia coli. Here, we report high-yield production (up to 230 mg/L from 3 mL cultures) of in-demand therapeutics such as IgG1-based Fc fusion proteins in the E. coli cytoplasm. However, the production of this drug class using the CyDisCo system faces bottlenecks related to redox heterogeneity during oxidative folding. Our investigations identified and addressed one of the major causes of redox heterogeneity during CyDisCo-based production of Fc fusion proteins, i.e., disulfide bond formation in the IgG1 CH3 domain. Here, we communicate that mutating the cysteines in the CH3 domain of target Fc fusion proteins allows their production in a homogeneous redox state in the cytoplasm of E. coli without compromising on yields and thermal stability.