Efficient Production of Fc Fusion Proteins in the Cytoplasm of Escherichia coli: Dissecting and Mitigating Redox Heterogeneity

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.

Design of an alternate antibody fragment format that can be produced in the cytoplasm of Escherichia coli

“…In addition, the Fab H 3 format is biologically active and shows a comparable binding affinity to the Fab format against the target antigen. Although the wild type IgG 1 C H 3 domain has been reported to exhibit redox heterogeneity when produced using CyDisCo in the cytoplasm of E. coli 14 , the suppression of homodimerization potentially slows down the rate of folding which allows the formation of disulfide bonds and thereby native folding. This is not the first report to use IgG C H 3 homodimers or heterodimers as scaffolds for the production of antibody formats, for e.g., scDb-C H 3 (KiH), Di-Diabody, Minibody, etc.…”

“…The cells were lysed by freeze thawing and as the proteins of interest contain a hexahistidine tag, they were purified with standard immobilized metal affinity chromatography (IMAC) using HisPur Cobalt Superflow Agarose (Thermo Scientific) resin under native conditions following clearance of the cell lysate by centrifugation (4000 rpm, 20 min, 4 °C). The protocol followed for Cobalt-IMAC based purification has been described in 14 .…”

“…This removes the limitations associated with the C H 1 domain in Fabs. The rationale behind using IgG 1 C H 3 domains is their high solubility and stability coupled with their remarkable dimerization propensity 14 , 15 . As heterodimerization is a critical attribute of Fabs, we modified the IgG 1 C H 3 domains using the previously established ‘Charge-to-charge swap design’ which allows efficient non-covalent heterodimerization of the C H 3 domains and allows the suppression of homodimerization though with a reduction in thermostability 16 .…”

Heterologous Expression of Difficult to Produce Proteins in Bacterial Systems

“…Taking a step further, an article published in the Special Issue has shown the potential of this system to produce mammalian extracellular matrix (ECM) proteins containing between 8 and 44 disulfide bonds, proving that this system has no upper limits for disulfide formation, making it possible to produce complex disulfide-bonded proteins in E. coli BL21(DE3). Moreover, Tungekar and Ruddock have recently noted that the CyDisCo system can also be used to recombinantly produce IgG1-based Fc fusion proteins [13].…”