The strategies biopharmaceutical manufacturers currently use to limit protein misfolding are complex, time-consuming, and generate low yields with only limited scalability. Covalent organic frameworks (COFs) offer a promising solution thanks to their rational design and tunability, which enables their internal interface interaction to be modulated precisely.
Recently published research identified the key COF elements that are integral to protein folding, and a one-step approach to modulate it.
A team led by senior authors Yao Chen, PhD, professor, Nankai University in Tianjin, China, and Wen Chen, PhD, professor, South China University of Technology, Guangzhou, China, identified pore size, hydrophobicity, pi-pi conjugation, and hydrogen bonding as being “critical to regulating protein conformation.”
With that evidence, the scientists developed a COF-directed protein refolding strategy that corrected misfolding and enabled recovery. That continuous, solid-phase system achieved a refolding yield of approximately 100% by gradually pumping lysozyme through the column. It was used for 30 cycles of refolding, elution, and regeneration.
“While our approach has demonstrated excellent refolding efficiency in model systems and shows strong potential for industrial use, it is currently at the experimental validation stage,” first author Jinbiao Guo, PhD candidate, Nankai University, tells GEN. “We believe that with further reengineering optimization and scale-up validation, it could become a key tool for biopharmaceutical manufacturing—particularly for improving protein refolding yields from inclusion bodies.”
For their experiments, the team designed a synthesized, mesoporous COF—NKCOF-122—and chose lysozyme as a test model because of its low molecular weight and well-known structure characterization.
Chen and colleagues found a correlation between COF pore size and the refolding yield of lysozyme. Specifically, NKCOF-122, with a 4.1 nm pore size that’s similar to that of lysozyme’s 4.5 nm, had the highest refolding rate, at 95%. When a larger, 5.4 nm pore size COF was used, the refolding yield dropped to about 53%. A pore size 1.8 nm smaller than lysozyme dropped the recovery yield to a mere 18%, “because lysozyme could only interact with the COF particle surface, rather than the COF pores,” they explained. The correlation between pore size and refolding yield continued when the protein was switched to glucose oxidase.
Furthermore, “Pi-to-pi conjugations promote protein refolding,” they reported, as do hydrophobic interactions. Hydrophobicity, they hypothesized, may “allow hydrophobic/hydrophilic groups in COFs to segregate partially folded proteins and reduce misfolding.” Additional factors affecting protein folding included hydrogen bonding and, to a small degree, the COF’s ability to adsorb the protein.
“Overall, the…results…verify the critical role of COFs’ internal environment in modulating protein folding, which may stabilize key intermediates and potentially lower the activation energy for correct folding,” the team noted.
These results also seem to apply to other proteins, based on results with trypsin, nattokinase, and papain. Each exhibited a high refolding efficiency exceeding 70% and, as hydrophobicity decreased, refolding efficiency also decreased. Therefore, this may be a more efficient purification and refolding process that has industrial protein manufacturing potential.
Next, Gao says, the scientists plan to “apply the platform to more complex proteins and real-world inclusion body systems, as well as develop a scalable column design that integrates seamlessly with existing manufacturing pipelines.” Customizing the COF microenvironment for different classes of proteins may also be explored.
The post Industrial-Scale Protein-Folding in One-Step Modulation appeared first on GEN - Genetic Engineering and Biotechnology News.
Recently published research identified the key COF elements that are integral to protein folding, and a one-step approach to modulate it.
A team led by senior authors Yao Chen, PhD, professor, Nankai University in Tianjin, China, and Wen Chen, PhD, professor, South China University of Technology, Guangzhou, China, identified pore size, hydrophobicity, pi-pi conjugation, and hydrogen bonding as being “critical to regulating protein conformation.”
With that evidence, the scientists developed a COF-directed protein refolding strategy that corrected misfolding and enabled recovery. That continuous, solid-phase system achieved a refolding yield of approximately 100% by gradually pumping lysozyme through the column. It was used for 30 cycles of refolding, elution, and regeneration.
“While our approach has demonstrated excellent refolding efficiency in model systems and shows strong potential for industrial use, it is currently at the experimental validation stage,” first author Jinbiao Guo, PhD candidate, Nankai University, tells GEN. “We believe that with further reengineering optimization and scale-up validation, it could become a key tool for biopharmaceutical manufacturing—particularly for improving protein refolding yields from inclusion bodies.”
Pore size affects refolding
For their experiments, the team designed a synthesized, mesoporous COF—NKCOF-122—and chose lysozyme as a test model because of its low molecular weight and well-known structure characterization.
Chen and colleagues found a correlation between COF pore size and the refolding yield of lysozyme. Specifically, NKCOF-122, with a 4.1 nm pore size that’s similar to that of lysozyme’s 4.5 nm, had the highest refolding rate, at 95%. When a larger, 5.4 nm pore size COF was used, the refolding yield dropped to about 53%. A pore size 1.8 nm smaller than lysozyme dropped the recovery yield to a mere 18%, “because lysozyme could only interact with the COF particle surface, rather than the COF pores,” they explained. The correlation between pore size and refolding yield continued when the protein was switched to glucose oxidase.
Furthermore, “Pi-to-pi conjugations promote protein refolding,” they reported, as do hydrophobic interactions. Hydrophobicity, they hypothesized, may “allow hydrophobic/hydrophilic groups in COFs to segregate partially folded proteins and reduce misfolding.” Additional factors affecting protein folding included hydrogen bonding and, to a small degree, the COF’s ability to adsorb the protein.
“Overall, the…results…verify the critical role of COFs’ internal environment in modulating protein folding, which may stabilize key intermediates and potentially lower the activation energy for correct folding,” the team noted.
These results also seem to apply to other proteins, based on results with trypsin, nattokinase, and papain. Each exhibited a high refolding efficiency exceeding 70% and, as hydrophobicity decreased, refolding efficiency also decreased. Therefore, this may be a more efficient purification and refolding process that has industrial protein manufacturing potential.
Next, Gao says, the scientists plan to “apply the platform to more complex proteins and real-world inclusion body systems, as well as develop a scalable column design that integrates seamlessly with existing manufacturing pipelines.” Customizing the COF microenvironment for different classes of proteins may also be explored.
The post Industrial-Scale Protein-Folding in One-Step Modulation appeared first on GEN - Genetic Engineering and Biotechnology News.