Insight

Recombinant proteins often form inactive inclusion body aggregates in prokaryotic expression systems such as Escherichia coli, and the refolding process constitutes the core bottleneck for obtaining functional proteins. This review systematically elaborates on the technical principles, research progress and applicable boundaries of five refolding strategies: dilution method, dialysis method, chromatographic method, microfluidic chip method and enzyme-assisted method. Analysis shows that traditional dilution and dialysis methods feature simple operation yet suffer from inherent limitations including restricted protein concentration and severe aggregation. Chromatographic techniques isolate protein molecules via solid-phase media, markedly improving refolding efficiency at high protein concentrations. Benefiting from precise microscale fluid control capability, microfluidic chips have emerged as a revolutionary platform for high-throughput condition optimization. The enzyme-assisted method specifically resolves molecular folding obstacles such as disulfide bond mismatching and proline isomerization. This article comprehensively introduces diverse refolding approaches for inclusion bodies.

The demand for recombinant proteins continues to grow in biomedicine, industrial catalysis and basic research. Owing to simple operation, low cost and high expression efficiency, E. coli has become the most commonly used recombinant protein expression system. However, under high-level expression stress, recombinant proteins frequently form inclusion bodies — inactive precipitates aggregated by misfolded polypeptide chains — due to insufficient intracellular folding capacity or environmental stress. The recovery process of inclusion bodies consists of four key steps: cell disruption, inclusion body isolation and washing, denaturant dissolution, and refolding. Among these steps, refolding poses the greatest challenge. Essentially, it refers to the process in which denatured polypeptide chains refold into correct three-dimensional structures and form native disulfide bonds as denaturants are gradually removed. The primary obstacle in this process is competitive aggregation, where partially folded intermediates form irreversible aggregates through hydrophobic interactions, resulting in an active protein recovery rate generally lower than 30%. Refolding efficiency is affected by multiple factors, including initial protein concentration, buffer conditions, temperature gradient and folding pathway kinetics. This paper aims to comprehensively summarize the technical principles and development of five refolding strategies, critically analyze their performance limitations, and provide theoretical guidance for strategy selection in different application scenarios.

As the most fundamental refolding strategy, dilution refolding involves rapidly diluting the denatured protein solution by 10 to 100-fold to instantaneously reduce the denaturant concentration below the critical threshold (usually less than 1–2 M urea or guanidine hydrochloride), thereby driving spontaneous protein folding. Optimized protocols include adding 0.4–1.0 M arginine to competitively bind exposed hydrophobic residues and inhibit aggregation, adopting reduced/oxidized glutathione (GSH/GSSG ratio: 1:5 ~ 1:1) to regulate correct disulfide bond pairing, and applying pulsed stepwise dilution to slow down aggregation kinetics. Nevertheless, this method has prominent drawbacks. Dilution leads to a volume expansion of over 50 times and drops the protein concentration below 0.1 mg/mL, which not only greatly increases subsequent concentration costs but also easily causes protein inactivation at low concentrations. In industrial scale-up, homogeneous mixing is difficult to achieve, resulting in poor batch-to-batch consistency. A typical study on lysozyme refolding shows an activity recovery rate of only 15%, highlighting the efficiency bottleneck of this method.

This technique employs semipermeable membranes with a molecular weight cutoff of 10–20 kDa to slowly reduce denaturant concentration via gradient diffusion, simulating the mild intracellular folding microenvironment. Technical improvements such as stepwise buffer replacement shorten the process duration to 12–24 hours, while reverse dialysis enhances mass transfer efficiency by reversing the solution arrangement. Its core advantage lies in maintaining a moderate protein concentration of 0.1–0.5 mg/mL, making it suitable for shear-sensitive proteins. However, it also has inherent shortcomings: the process lasts 24–72 hours with long time consumption; membrane surface adsorption causes an irreversible protein loss of 20–30%; the control accuracy of denaturant gradient decline is limited; and membrane costs rise significantly during large-scale production. For the same lysozyme system, although the recovery rate can be increased to 25%, the processing time is more than tripled.

The core breakthrough of chromatographic technology lies in isolating denatured protein molecules through solid-phase media, effectively suppressing intermolecular collision and aggregation. It elevates the initial protein concentration to 1–5 mg/mL and realizes integrated refolding and purification. Based on action mechanisms, chromatography-mediated refolding is classified into four categories. Size-exclusion chromatography separates proteins by molecular weight, with protein folding completed in the mobile phase; it is applicable to proteins below 50 kDa but limited by low loading capacity. Ion-exchange chromatography relies on electrostatic interactions to achieve refolding via salt concentration or pH gradient elution, featuring high loading capacity and efficient aggregate removal. A typical case shows that the recovery rate of proinsulin on DEAE (Diethylaminoethyl) media reaches 60%. Hydrophobic interaction chromatography triggers protein folding through high-salt hydrophobic adsorption and low-salt elution, exhibiting prominent advantages for hydrophobic membrane protein fragments; the refolding rate of single-domain antibodies on phenyl sepharose columns reaches 75%. Affinity chromatography achieves targeted refolding via specific binding of tags such as His-Tag, with outstanding selectivity yet high cost. Overall, this technology is constrained by high equipment investment, complicated method development and medium adsorption risks.

Microfluidic platforms enable precise manipulation of refolding environments via microscale fluid effects, with three major innovative strengths. First, laminar diffusion mixing completes denaturant dilution or buffer exchange within 100 milliseconds, avoiding the formation of aggregation-prone folding intermediates. Second, picoliter-scale reaction volumes require only microgram-level protein per test, drastically reducing consumption of precious samples. In addition, parallel channel design supports simultaneous screening of 96 sets of conditions on a single chip. Mainstream microfluidic platforms include Y/T-type continuous-flow micromixers and oil-water segmented droplet microreactor systems. In the refolding study of interferon-β, after optimization with 0.6 M arginine and pH 8.5, the recovery rate surged from 32% via dialysis to 68%. Current technical bottlenecks include complex chip fabrication, channel blockage risks and difficulties in scaling up from microscale to industrial production; microfluidics mainly serves as a front-end optimization tool for macroscopic process development.

Targeting molecular-scale folding obstacles such as disulfide bond mismatching and proline cis-trans isomerization, key enzymatic agents mainly include Protein Disulfide Isomerase (PDI) and Peptidyl Prolyl cis-trans Isomerase (PPIase). PDI catalyzes correct disulfide bond pairing through triple activities of oxidation, reduction and isomerization, while PPIase accelerates the rate-limiting step of X-Pro peptide bond cis-trans isomerization. Common application strategies include directly adding 0.1–1 μM PDI and 0.5–2 μM PPIase to refolding buffers, immobilizing enzymes on chromatographic media for continuous catalysis, and adopting thioredoxin fusion expression to utilize its PDI-like activity. The refolding rate of antibody fragments containing 8 pairs of disulfide bonds can be increased from less than 5% to 40%. However, the high cost of enzyme preparations restricts large-scale application, which requires improving enzyme stability and catalytic efficiency via protein engineering modification.

Common inclusion body refolding and recovery strategies cover dilution method, dialysis method, microfluidic method, chromatographic method and enzyme-assisted method. These methods differ significantly in activity recovery rate, tolerable protein concentration, time efficiency and cost. For strategy selection: microfluidics is preferred for microscale condition screening; chromatography is optimal for high-concentration processing requirements; enzyme-assisted methods are adopted to enhance refolding efficiency of proteins with multiple disulfide bonds; optimized dilution or dialysis formulas are suitable for cost-sensitive projects. Although easy-to-operate dilution and dialysis methods are widely used in preliminary screening, their low recovery rate and time-consuming nature limit industrial application. Chromatography dominates large-scale high-concentration refolding by virtue of solid-phase molecular isolation. Microfluidic technology enables rapid screening with millisecond-level precise control, while enzyme-assisted strategies target specific molecular folding barriers. Future development lies in the integration of computational modeling, microfluidic screening, chromatographic scale-up and enzymatic efficiency enhancement, aiming to break production bottlenecks of high-value therapeutic proteins such as CAR-T (Chimeric Antigen Receptor T-cell) cytokines and complex antibodies, and promote overall technical efficiency and industrial upgrading.