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. 2026 Feb 4;6(2):1197–1205. doi: 10.1021/jacsau.5c01572

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

Shunpei Iwamoto †, Yuya Nishizawa †, Hayato Yokose ‡,§, Osamu Kanie ∥, Yosuke Okamura ‡,§, Takahiro Muraoka ⊥,*, Kenta Arai †,#,*
PMCID: PMC12933366  PMID: 41755826

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.

Keywords: heterogeneous catalysis, selenium, protein disulfide isomerase, protein folding, enzyme models, sustainable chemistry, materials science

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Introduction

Protein folding is a fundamental process by which polypeptides acquire their functional three-dimensional structures. , The failure of protein folding is associated with various diseases, such as neurodegeneration, diabetes, and atherosclerosis. − Therefore, cells have mechanisms to assist nascent polypeptides in folding correctly. − These mechanisms include molecular chaperones that ensure protein quality by transiently capturing unfolded proteins and facilitating structural maturation. Even in vitro, accurate folding is critical in areas such as chemical protein synthesis, enzyme engineering, and biopharmaceutical production, directly affecting research outcomes and manufacturing efficiency. −

Many secretory and membrane proteins require the precise formation of disulfide (SS) bonds between specific pairs of cysteine (Cys) residues to attain their native structures. , This oxidative folding is catalyzed by protein disulfide isomerase (PDI), − which promotes both SS formation and isomerization (Figure a, top). PDI is composed of four thioredoxin-like domains arranged in a U-shaped conformation with a hydrophobic cavity that transiently captures folding intermediates, thereby accelerating folding and suppressing undesired aggregation (Figure a, bottom).

1.

1

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Oxidative protein folding catalyzed by natural and artificial systems. (a) Schematic illustration of oxidative folding by PDI via SS formation and isomerization at redox-active sites. (b) Conventional workflow of oxidative folding with single-use enzymes and reagents. (c) Design concept of a “solid chaperone” equipped with a diselenide catalyst that promotes SS formation and isomerization. (d) Workflow of recyclable oxidative folding using a solid chaperone.

Thus, PDI acts not only as an oxidoreductase but also as a molecular chaperone. − Although PDI can accelerate folding in vitro and enhance productivity, − its industrial application is limited by high preparation costs, high-performance liquid chromatography (HPLC) purification requirements, and frequent denaturation during recovery, which prevents reuse (Figure b).

Artificial oxidative folding systems have been explored as alternative platforms. − Small-molecule promoters, often thiol (SH)/SS compounds inspired by the redox-active site of PDI, catalyze both SS formation and isomerization in Cys-rich proteins through PDI-like mechanisms (Figure a, top). − Furthermore, selenol (SeH)/diselenide (SeSe) molecules have superior physicochemical properties and higher catalytic activity than their sulfur analogs, highlighting their potential as efficient folding promoters. −

However, while a few small molecules can mimic chaperone-like activity via hydrophobic or electrostatic interactions, , rationally designing artificial low-molecular-weight chaperones remains highly challenging. Additionally, the requirement of the postreaction separation of folding promoters continues to be a major bottleneck, especially in pharmaceutical manufacturing, where strict purity standards are indispensable. Moreover, practical constraints, such as the high synthetic costs and limited reusability of these promoters, present additional hurdles to their broader application (Figure b).

Overall, an ideal oxidative folding catalyst should meet four requirements:

  • i.

    reusability,

  • ii.

    separation without HPLC,

  • iii.

    chaperone-like function, and

  • iv.

    high redox activity.

Herein, we report a heterogeneous catalyst that fulfills all four criteria. Incorporating SeSe motifs into a polystyrene resin that captures immature proteins via hydrophobic interactions created a solid material with dual catalytic and chaperone-like functions (Figure c). The catalyst promoted oxidative folding while suppressing protein aggregation and was readily recovered by filtration for repeated use (Figure d). These findings support the adoption of the “solid chaperone” as a fundamental platform for oxidative protein foldingone that not only reproduces the dual functions of PDI but also provides a reusable, scalable, and purification-free alternative to conventional solution-phase folding systems.

Results and Discussion

Preparation of the “Solid Chaperone”

To embody the “solid chaperone” concept, we prepared a redox-active solid material. As the redox motif, we selected (S)-1,2-diselenan-4-amine (1), synthesized from inexpensive aspartic acid in seven steps with an overall yield of 47%. Compound 1 was covalently attached to a PEGylated polystyrene-based carboxy resin (2, NovaSyn TG, φ ≈ 130 μm, substitution: 0.27 mmol/g; Figures a and S1) through amide bond formation using DIPEA and PyBOP, affording resin 3 (Reaction A in Figure b). Owing to its PEGylation, resin 2 swells well in both organic and aqueous media, making it suitable for evaluating oxidative folding reactions in a buffer. Scanning electron microscopy (SEM)-energy dispersive X-ray spectroscopy (EDS) analysis confirmed the presence of Se atoms on the resin surface (Figure c–e). Ellman’s assay further quantified the SeSe loading as 17.2 ± 3%, supporting the successful immobilization of compound 1 (Reaction B in Figure b).

2.

2

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Preparation and characterization of the redox-active solid chaperone. (a) Scanning electron microscopy (SEM) image of the redox-inactive polystyrene-based carboxy resin (2, scale bar = 10 μm). Samples were Pt-coated before imaging. (b) Preparation of resin 3. (Reaction A) Immobilization of cyclic diselenide 1 onto resin 2. (Reaction B) Quantification of SeSe moieties using Ellman’s assay (see Supporting Information). (c) SEM image of resin 3 (scale bar = 1 μm). (d) Energy-dispersive X-ray spectroscopy (EDS) elemental mapping showing Se distribution in resin 3. (e) EDS spectrum of the region highlighted in (c).

Catalytic Performance and Substrate Scope of the “Solid Chaperone”

Next, we evaluated the catalytic performance of resin 3 in oxidative folding using model proteins. As the first model, we employed a reduced form of oxytocin (ROxy), a short peptide hormone containing one SS bond (Figure a). ROxy was incubated with resin 3 (25 mol% as SeSe units) in buffer (pH 7.5) at 30 °C, and the reaction progress was monitored using HPLC after quenching (Figures d and S2). For comparison, a parallel reaction with resin 2, which lacks the SeSe modification, was performed. After 60 min, the peak corresponding to ROxy remained detectable in the chromatogram, whereas resin 3 completely converted ROxy into the native form (NOxy) via SS formation. This transformation proceeded quantitatively with only one-quarter of the stoichiometric amount, clearly demonstrating the catalytic nature of the system (vide infra). Furthermore, the homogeneous SeSe catalyst (1) showed no significant activity under the same conditions (Figure g). These results suggest that the enhanced SS formation by resin 3 arises from amplification of the intrinsic activity of catalyst 1 by the polystyrene support.

3.

3

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Oxidative folding of model proteins using resin 3. (a–c) Structures of oxytocin (a), endothelin-1 (b), and hirudin (c). (d–f) HPLC chromatograms obtained from oxidative folding of the reduced state of oxytocin (ROxy, d), endothelin-1 (RET1, e), and hirudin (RHir, f). The assignment and identification of NOxy have been previously reported. NET1 and NHir were identified by direct comparison of HPLC retention times with authentic standards and further confirmed by MS analysis (Figure S3). (g–i) Time courses of native formation for oxytocin (NOxy, g), endothelin-1 (NET1, h), and hirudin (NHir, i), monitored using HPLC. Reaction conditions: ROxy (100 nmol in 1.0 mL buffer, pH 7.5), RET1 (40 nmol in 0.5 mL buffer, pH 8.0), and RHir (80 nmol in 1.0 mL buffer, pH 8.0) were incubated with 1, 2, or 3 under aerobic conditions with shaking (30 °C for ROxy; 25 °C for RET1 and RHir). Catalyst loadings: 25, 40, and 80 nmol as SeSe units for ROxy, RET1, and RHir, respectively. Data represent mean ± standard error of the mean (s.e.m.) (n = 3).

To further examine the catalytic properties of resin 3, we conducted folding experiments using reduced endothelin-1 (RET1), a vasoconstrictor peptide containing two SS bonds, as a model substrate (Figure b). Reaction progress was monitored using HPLC after quenching (Figures e and S4). When either homogeneous catalyst 1 or resin 2 was used, RET1 remained the major component even after 60 min, showing little folding promotion. In contrast, resin 3 (50 mol% as SeSe units) rapidly induced the native state (NET1), reaching ∼78% yield within 60 min and plateauing at 93% after 120 min (Figure h). Control experiments using separately added catalyst 1 and resin 2 showed no significant enhancement (Figure S4), indicating that the superior activity of resin 3 arises from the synergistic effect between the immobilized SeSe moiety and the polystyrene support. In addition, folding of RET1 was examined in a conventional GSH/GSSG (1.0 mM/0.2 mM) redox buffer. Under these conditions, the folding rate was clearly slower than that achieved with resin 3 (Figure S4e,f), despite the use of GSH/GSSG in large excess relative to the substrate (80 μM). This comparison highlights the high folding-promoting efficiency of resin 3 operating at catalytic loading.

To evaluate the applicability of this system to proteins with more complex SS-bonding patterns, we performed similar folding experiments using the reduced form of hirudin (RHir), a blood anticoagulant protein with three SS bonds (Figure c). Using resin 3 (33 mol% as SeSe units), RHir was rapidly converted into partially oxidized intermediates soon after reaction initiation (Figure S5), and after 300 min, the native state (NHir) was detected as the major product (Figure f). In contrast, with catalyst 1 or resin 2, residual RHir persisted, and NHir formation was negligible (Figures f and S5).

Catalytic Activity for SS Formation and Isomerization on the Resin Surface

In a PDI-like manner (Figure a, top), cyclic SeSe moieties catalyzed SS formation through intermolecular SH–SeSe exchange (Figure ). The resulting diselenol (4), owing to its high nucleophilicity, promoted SS isomerization, ultimately yielding native proteins with the correct SS pattern. A portion of diselenol was reoxidized by O2, enabling complete SS formation even when resin is used at substoichiometric amounts.

4.

4

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Proposed catalytic cycles for oxidative folding on resin.

In the case of hirudin, resin 3 markedly accelerated folding; however, the folding yield was limited to 65% (Figure i), likely because the formation of active diselenol species was insufficient to promote effective SS isomerization. This limitation was overcome by adding reduced glutathione (GSH), which shifted the SeSe 3/diselenol 4 equilibrium toward the diselenol form, thereby facilitating SS isomerization (Figure ). Under these conditions, folding proceeded effectively, affording NHir in 99% yield (Figure S6). Nevertheless, heterogeneous catalysts rarely outperform their homogeneous counterparts; therefore, the striking activity of resin 3 cannot be explained by the surface redox chemistry alone.

Chaperone-Like Function on the Resin Surface

The remarkable rate enhancement observed with resin 3 is likely attributable to the transient localization of structurally immature proteins on the resin surface via hydrophobic interactions, which facilitates their contact with the SeSe sites. To test this hypothesis, phenol was added to inhibit such interactions competitively, and the oxidative folding of hirudin with resin 3 was examined. As a result, a significant decrease in the reaction rate was observed, whereas phenol addition had no effect when using homogeneous catalyst 1 (Figures i and S5). Combined with the finding that separate addition of catalyst 1 and resin 2 showed no significant catalytic enhancement (Figure S4), these results strongly suggest that the superior activity of resin 3 arises from the polystyrene support, which provides a favorable reaction environment for oxidative folding.

To further support this hypothesis, we evaluated the protein adsorption capacity of the polystyrene resin (2). Reduced (RHEL) and native (NHEL) forms of hen egg-white lysozyme (143 μg/mL) were employed as model proteins (Figure a). After mixing with resin 2 (50 mg/mL), protein adsorption was quantified using the bicinchoninic acid (BCA) assay. RHEL showed approximately 2.5-fold higher adsorption than NHEL on resin 2 (Figure b), indicating that the polystyrene support preferentially binds structurally immature proteins and contributes to the enhanced catalytic activity of the SeSe moiety. In oxidative folding experiments, the protein elution amounts estimated from HPLC were identical regardless of the catalyst employed. This observation indicates that the interaction between the substrate proteins and the resin is of low affinity, ensuring that the proteins do not remain adsorbed on the resin surface under catalytic conditions.

5.

5

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Oxidative folding promotion by resin 3 via its chaperone-like activity. (a) Oxidative folding of RHEL into NHEL. (b) Protein adsorption onto polystyrene-based resin 2. RHEL or NHEL (143 μg/mL) was incubated with resin 2 (50 mg/mL) for 3 h in buffer (1.0 mL, pH 7.5). Adsorbed protein was quantified using the bicinchoninic acid (BCA) assay (see Supporting Information). (c–e) HPLC chromatograms obtained from oxidative folding of HEL at a high concentration. RHEL (100 nmol) and a catalyst (1, 2, or 3; 100 nmol) were mixed in buffer (1.0 mL, pH 7.5) containing 2 M urea and incubated at 37 °C under aerobic conditions with shaking. NHEL was identified by direct comparison of HPLC retention times with authentic standards and further confirmed by MS analysis (Figure S3). (f) Time course of NHEL yield. Conditions were the same as in (c–e). Yields were estimated from HPLC data using a calibration curve. (g) Photograph of sample solutions obtained after oxidative folding of RHEL for 6 h. Data in (b) and (f) represent mean ± s.e.m. (n = 3).

This unique property of polystyrene suggests that resin 3 exhibited chaperone-like functionality during oxidative folding. At high substrate concentrations, where aggregation competes with folding, the chaperone-like activity of resin 3 is presumed to suppress aggregation and promote correct folding. When oxidative folding of RHEL was performed at 100 μM (ca. 1.4 mg/mL), reactions with resin 2 showed reduced protein elution in HPLC due to aggregation (Figure d,g), and only negligible levels of NHEL were formed (Figure f). When catalyst 1 was added, RHEL was slowly converted into SS intermediates and then yielded NHEL (Figure e). However, aggregation occurred in parallel (Figure g), and the overall folding yield remained low (ca. 20%) (Figure f). In contrast, resin 3 effectively suppressed aggregation (Figure g), rapidly converted RHEL into SS intermediates (Figure c), and afforded NHEL in ∼50% yield (Figure f). These results demonstrate that resin 3 guides the folding pathway toward the native state through its chaperone-like activity.

Recyclable Oxidative Folding Using the “Solid Chaperone”

In conventional homogeneous systems, the removal of enzymes and catalysts often requires HPLC, which hinders scaled-up and industrial applications. In contrast, resin 3 can be readily recovered using simple filtration, achieving HPLC-free recovery (Figure a). The recovered resin exhibited excellent physical stability, with no visible deterioration observed after reuse (Figures b and S7). Furthermore, in reuse experiments, no significant loss of catalytic activity was observed even after four consecutive runs with endothelin-1 as a substrate (Figures c and S8), clearly demonstrating the robust reusability of resin 3. By enabling HPLC-free recovery and repeated use, the “solid chaperone” offers a robust platform for process intensification in protein production.

6.

6

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Reusability of resin 3 using endothelin-1 as a model protein. (a) Workflow of recyclable oxidative folding. (b) SEM images of resin 3 before and after four uses. (c) Time course of NET1 formation over repeated cycles. Reaction conditions: RET1 (32 nmol) and resin 3 (32 nmol as SeSe units) in buffer (400 μL, pH 8.0) were incubated at 25 °C under aerobic conditions with shaking.

Conclusion

We developed a solid-phase catalyst for oxidative folding by covalently immobilizing a cyclic diselenide on a polystyrene resin. This “solid chaperone” promoted the formation of native proteins in up to 99% yield, suppressed aggregation even at high substrate concentrations, and was readily recovered and reused without the loss of catalytic activity. These features fulfill all four fundamental criteria for an ideal oxidative folding catalyst: (i) reusability, (ii) HPLC-free isolation, (iii) chaperone-like aggregation suppression, and (iv) high redox efficiency (Figure d).

Importantly, this work demonstrates that the dual functions of PDIredox catalysis and the transient capture of folding intermediatescan not only be mimicked but can also be extended beyond the intrinsic limitations of enzymatic and homogeneous solution-phase systems. In contrast to natural PDI, which is confined to fragile, single-use operation, and purification-dependent workflows (Figure b), this heterogeneous catalyst offers operational stability and process flexibility suited to scalable protein manufacturing.

In summary, by rationally re-engineering complex biological folding functions into a robust polymer support, this study establishes a materials-based framework for oxidative protein folding and lays the foundation for recyclable, enzyme-free folding systems. As a proof of concept, it also highlights the need for further studies on substrate scope, mechanistic insight, and long-term performance under industrially relevant conditions, paving the way for sustainable and scalable alternatives to enzyme-dependent folding systems.

Supplementary Material

au5c01572_si_001.pdf (20.5MB, pdf)

Acknowledgments

We thank the Biotechnology Laboratory, Japan Energy Co., Ltd., for kindly providing the recombinant hirudin variant (CX397). This work was supported by the Japan Society for the Promotion of Science (JSPS) [KAKENHI: grant number 23K04933 (KA)]. This study was also supported in part by a grant from the Kanagawa Prefecture Government of Japan (KA).

Glossary

Abbreviations

Cys

cysteine

DIPEA

N,N-diisopropylethylamine

EDS

energy dispersive X-ray spectroscopy

GSH

reduced glutathione

HPLC

high-performance liquid chromatography

NET1

native form of endothelin-1

NHEL

native form of hen egg-white lysozyme

NHir

native form of hirudin

NOxy

native form of oxytocin

PDI

protein disulfide isomerase

PyBOP

benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate

RET1

reduced form of endothelin-1

RHEL

reduced form of hen egg-white lysozyme

RHir

reduced form of hirudin

ROxy

reduced form of oxytocin

s.e.m.

standard error of the mean

SeH

selenol

SEM

scanning electron microscopy

SeSe

diselenide

SH

thiol

SS

disulfide.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.5c01572.

  • Materials and Methods: detailed procedures for resin synthesis and oxidative folding assays. Elemental analyses of resins 2 and 3 (Figures S1, S7). Identification of the native state by HPLC and mass spectrometry (Figure S3). HPLC chromatograms for oxidative folding of oxytocin, endothelin-1, and hirudin (Figures S2, S4, and S5). Oxidative folding of hirudin in the presence of GSH (Figure S6). HPLC chromatograms from the reusability assessment of resin 3 (Figure S8) (PDF)

K.A. conceived the research concept and initiated the project. S.I. and Y.N. synthesized the redox-active resin (“solid chaperone”) and evaluated its catalytic performance in oxidative protein folding. S.I., H.Y., and Y.O. assessed the protein adsorption properties of the resin. O.K., T.M., and K.A. supervised the research. S.I., T.M., and K.A. wrote the manuscript with input from all authors. All authors have approved the final version of the manuscript. CRediT: Shunpei Iwamoto data curation, formal analysis, investigation, validation, writing - review & editing; Yuya Nishizawa data curation, formal analysis, investigation; Hayato Yokose investigation; Osamu Kanie supervision; Yosuke Okamura formal analysis, investigation, validation; Takahiro Muraoka supervision, writing - original draft, writing - review & editing; Kenta Arai conceptualization, supervision, writing - original draft, writing - review & editing.

This work was supported by JSPS KAKENHI (23K04933) and by a grant from the Kanagawa Prefecture Government of Japan.

The authors declare no competing financial interest.

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