Beyond Folding Enzymes: A Redox-Active "Solid Chaperone" Unlocks Recyclable, HPLC-Free Oxidative Protein Folding

超越折叠酶:一种氧化还原活性“固体伴侣”解锁可回收、无需高效液相色谱法的氧化蛋白质折叠

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Abstract

Oxidative protein folding, which is critical to proteins achieving their functional structures, is catalyzed in cells by protein disulfide isomerase (PDI)an enzyme that couples redox catalysis with the transient capture of folding intermediates to promote native disulfide formation while preventing aggregation. Although PDI improves oxidative folding in both chemically synthesized and recombinantly produced proteins, its use is restricted to homogeneous systems, limiting reusability and operational robustness. Artificial PDI mimics have advanced in vitro folding; however, no system has yet combined sufficient redox activity for native disulfide formation with a folding environment that suppresses aggregation, nor demonstrated true reusability. Here, we introduce a polymer-based "solid chaperone" that realizes PDI-like dual activity on an abiotic surface, achieving what natural PDI cannot: recyclable, HPLC-free oxidative folding without the stability and single-use limitations of enzymes. The covalent immobilization of cyclic diselenide onto polystyrene beads yields a redox-active and hydrophobic interface that transiently captures unfolded proteins, catalyzes both disulfide bond formation and isomerization, and suppresses aggregation even at high substrate concentrations. This solid-phase catalyst outperformed its homogeneous counterpart, producing native peptides and proteins in up to 99% yield and retaining full activity over multiple reuse cycles. These results demonstrate that complex biological folding functions, once confined to fragile enzymes, can be re-engineered into durable polymeric materials. This solid-phase strategy not only enables recyclable oxidative folding but also establishes a paradigm for translating enzymatic behavior into scalable synthetic systems with industrial potential.

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