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Biomanufacturing

High Throughput/Scalable Production

2-Stage Dynamic Control Enables Robust, High-Titer, Cytoplasmic Expression of Nanobodies in E. coli

Friday, October 13, 2023
2:30 PM – 2:45 PM PDT
Location: 332 (Center, Level 3)
Introduction:: Nanobodies (VHHs) are useful therapeutic and diagnostic agents with some advantages over conventional antibodies and their fragments. VHHs, derived from the variable domain of heavy-chain-only antibodies in the Camelidae family, are small proteins capable of binding to antigens with a single domain, which provides advantages with respect to diffusivity, epitope binding, and stability. VHHs have been approved by the FDA, and there is growing interest in low-cost production platforms. However, cytoplasmic expression of nanobodies in E. coli presents a challenge due to the reductive nature of the cytoplasm. VHHs contain at least 1 conserved disulfide bond, and reduction of this bond results in improper folding and insoluble aggregation. Previous approaches to promote disulfide bond formation in the E. coli cytoplasm have resulted in negative impacts on growth and protein expression. Strains have been engineered to constitutively increase the cytoplasmic oxidative potential by deleting key enzymes in reducing pathways. Additionally, overexpression of a disulfide bond isomerase (dsbC) and disulfide bond oxidase (erv1p) have improved soluble expression of proteins containing disulfide bonds. Specifically, erv1p catalyzes cysteine oxidation and dsbC isomerizes disulfide bonds to improve correct folding. We report improved cytoplasmic VHH expression with two-stage dynamic control over the redox state, disulfide oxidase and isomerase activities. This approach separates growth and nanobody production into two phases, preserving growth robustness while achieving high-titer nanobody expression. Finally, we integrated dynamic expression of enzymatic components needed for cellular autolysis, resulting in rapid autolysis and nanobody purification.

Materials and Methods:: Dynamic control separates growth and recombinant protein expression. In the first phase, the E. coli metabolism remains unaltered and cells grow normally. The nanobody is placed under the control of a phoB promoter, which is activated under low phosphate conditions. Phosphate concentration in the media is the trigger to induce VHH expression. The media is formulated for autoinduction coincident with the phosphate depleted stationary phase. Not only does phosphate depletion “turn-on” VHH expression, it is also a trigger to simultaneously “turn off” and “turn-on” key enzymes controlling cytoplasmic redox state and nanobody folding. Engineered strains are assayed with a redox sensitive GFP. The strain with the highest cytoplasmic oxidation was modified for deletion of glutathione reductase (gor), dynamic reduction in thioredoxin reductase (trxB) levels, overexpression of dsbC, evr1p, and the enzymatic components needed for autolysis, again upon entry into the stationary phase. Robustness studies were performed with this strain to characterize growth and protein expression. Nanobody expression was quantified in shake flask studies with SDS-PAGE and densitometry. Lastly, purified VHHs were used to confirm VHH functionality.

Results, Conclusions, and Discussions:: Dynamic control strains have improved growth and protein expression compared to E. coli strains engineered via constitutive approaches. Additionally in previous studies, the impact of the individual “modules'', ie. reductase deletions versus chaperone overexpression, has not been well understood. Dynamic control of individual modules (reductase control, dsbC, and erv1p), was used to understand the impact of each component on VHH solubility. Our results showed that the combination of all three components has a synergistic effect leading to improved soluble VHH expression. After this initial confirmation, we tested expression of 16 VHHs with 1-2 disulfide bonds. 80% of tested VHH proteins were soluble while 68% exceeded the upper limit of periplasmic yield (100mg/L), and 50% surpassed the highest reported yield for cytoplasmic expression (200mg/L). The highest VHH yield achieved was >700mg/L in a shake flask. In comparison, in the control 56% of the sequences were soluble and only 12% had yields exceeding periplasmic expression. Two VHHs from the library, both binding GFP and minimizing or enhancing fluorescence, were purified after autolysis and activity was confirmed based on a GFP binding assay. This expression platform offers a tightly controlled, robust and scalable approach for VHH expression and purification.

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