Insight

Efficient expression of recombinant proteins in Escherichia coli relies on inhibiting non-functional aggregation of nascent polypeptide chains and facilitating their directional folding into native conformations. Multi-dimensional collaborative optimization strategies are required to achieve this goal. Firstly, abnormal aggregation of folding intermediates can be effectively suppressed by introducing molecular chaperones to shield hydrophobic patches, adding detergents to reduce surface tension, or expressing complex subunits to maintain structural stability. Secondly, folding enzymes that catalyze disulfide bond formation and isomerases that rectify erroneous conformations can accelerate the folding process and prevent the accumulation of metastable configurations.

For disulfide bond-dependent proteins, the formation of Cys-Cys bridges is strictly regulated by the redox potential of subcellular compartments. Eukaryotic systems rely on the oxidative environment of the endoplasmic reticulum, while E. coli achieves equivalent functions via the periplasmic space. An appropriate microenvironment for disulfide bond formation can be established by targeting eukaryotic proteins to the bacterial periplasm through signal peptide engineering or modifying the cytoplasmic redox system. For formed inclusion bodies, chaotropic agents can be used for solubilization to obtain monodispersed polypeptide chains, followed by gradient dialysis combined with oxidants for gradual renaturation to restore native structures. Current technical routes demonstrate that the integration of signal peptide targeting, synergistic action of oxidative folding cofactors and in vitro renaturation processes can markedly enhance the functional expression level of disulfide bond-dependent recombinant proteins.

The most straightforward approach to harvest folded disulfide bond-dependent recombinant proteins using E. coli is to direct translated polypeptides into the bacterial periplasm. This method is physiologically rational: compared with the cytoplasm, the periplasm constitutes an oxidative compartment harboring enzymes that catalyze disulfide bond formation and isomerization, as well as specific chaperones and folding enzymes. Nevertheless, the necessity of translocating nascent polypeptides across the inner membrane introduces a rate-limiting step. Given the limited number of available translocation portals to the periplasm, metastable precursors tend to accumulate in the cytoplasm.

Optimizing the expression of disulfide bond-dependent proteins in E. coli requires dual-dimensional regulation covering translational modulation and secretory targeting. Modification of the Shine-Dalgarno sequence and its upstream and downstream translation initiation regions enables fine-tuning of translation rates, thereby alleviating the inhibition of secretory efficiency caused by cytoplasmic overcrowding. Meanwhile, synergistic optimization of parameters including promoter types, plasmid replication origins and culture medium components can balance the contradiction between protein expression rate and folding capacity.

In terms of secretory pathways, the selection of leader peptides exerts a decisive effect on the translocation efficiency of target proteins. Both natural signal peptides such as OmpA and PelB and synthetic signal peptides have been widely applied. Their core mechanism lies in directing unfolded polypeptides to the oxidative periplasmic space via post-translational translocation through the Sec system or co-translational translocation through the SRP system. Notably, the compatibility between leader peptides and chaperone proteins also affects secretory efficiency. The systematic integration of the above strategies provides a theoretical framework and technical approaches to break through cytoplasmic folding restrictions and realize efficient secretion of disulfide bond-dependent proteins.

Disulfide bond formation in the E. coli periplasm is precisely regulated by the Dsb system. As a core oxidase, DsbA catalyzes disulfide bond formation in nascent peptide chains with the assistance of DsbB, and its hydrophobic interface also exhibits molecular chaperone activity. However, excessive oxidation easily leads to incorrect disulfide linkage. Under such circumstances, isomerases DsbC and DsbG recognize misfolded proteins via their V-shaped domains and catalyze disulfide bond rearrangement while maintaining a reduced state supported by DsbD.

Functional complementation studies confirm that co-expression of DsbA and DsbC is critical for the folding of complex proteins with discontinuous disulfide bonds. DsbA preferentially mediates oxidation of adjacent cysteines, whereas DsbC specializes in repairing long-range disulfide bonds. Cross-species research has verified that mammalian isomerases can functionally substitute their bacterial counterparts in E. coli, offering novel strategies for eukaryotic protein expression. The rational combination of Dsb family proteins serves as a key measure to improve the correct folding rate of recombinant proteins, and customized synergistic networks of oxidative isomerases should be designed according to the disulfide bond topological characteristics of target proteins.

Protein folding in the E. coli periplasm is coordinately regulated by molecular chaperones and folding enzymes. SurA and FkpA elevate the correct folding efficiency of complex structural proteins such as retinol-binding proteins by stabilizing folding intermediates, while DegP maintains the solubility of aggregation-prone proteins through dual functions as a chaperone and protease. Peptidyl-prolyl isomerases directly participate in the rate-limiting step of antibody folding, and their co-expression as fusion tags can greatly increase the yield of functional antibodies.

The auxiliary mechanism of cytoplasmic chaperones in periplasmic folding remains poorly elucidated, which may function via non-specific stabilization of unfolded intermediates. As a natural secretory chaperone, SecB specifically recognizes nonapeptide motifs containing aromatic and basic residues, yet it possesses limited binding capacity to non-specific substrates. This defect can be compensated by overexpressing other chaperones, which prevent polypeptide aggregation through broad-spectrum binding activity.

In addition, supplementation of chemical chaperones optimizes the folding microenvironment to facilitate correct protein folding. Combined application of reduced glutathione and DsbC overexpression remarkably enhances the stability of disulfide bond-dependent proteins. These multi-dimensional interventions targeting periplasmic and cytoplasmic folding networks establish a systematic solution for high-efficiency expression of recombinant proteins.

The fusion protein strategy applied in E. coli periplasmic expression significantly boosts recombinant protein yields by stabilizing folding intermediates, yet it inevitably has inherent limitations. For instance, maltose-binding protein fusion increases the production of single-chain variable fragments (scFv) but results in antibody inactivation. In contrast, alkaline phosphatase fusion not only enhances protein yield but also improves antibody affinity through dimerization and enables enzyme-linked immunoassays.

Multiple fusion systems including protein A domains and glutathione S-transferase are widely utilized for the soluble expression and purification of complex proteins such as scFv and coagulation factors. It is noteworthy that certain fusion designs mainly expand protein functional properties rather than optimizing folding efficiency.

Apart from the Sec and SRP systems, the twin-arginine translocation (Tat) pathway mediates the transport of fully folded intact proteins across membranes, endowing it with unique advantages in recombinant protein expression. The Tat system is encoded by the tatABC operon, and its overexpression can substantially improve the transmembrane transport efficiency of folded substrates such as fluorescent reporter proteins.

Key chaperones including DnaK, SlyD and TorD enhance the efficacy of the Tat pathway through distinct mechanisms. DnaK not only promotes correct substrate folding but also temporarily sequesters immature proteins by recognizing Tat leader peptide sequences to avoid premature translocation. This mechanism is particularly vital for the transport of complex proteins like scFv antibodies. After complete folding in cytoplasm with optimized reducing conditions, target proteins can be efficiently exported to the periplasm via the Tat system.

Although chaperones such as FkpA in the E. coli periplasm inhibit inclusion body formation, overexpression of heterologous proteins still tends to trigger protein aggregation. Notably, there are research blind spots in the study of periplasmic inclusion bodies: conventional experiments often neglect stepwise cellular lysis analysis, which may lead to underestimation of their actual occurrence frequency.

To simplify downstream purification, researchers have developed strategies to induce targeted secretion of periplasmic proteins into culture media. In addition to the traditional osmotic shock method, the Kil protein expression system achieves controlled outer membrane lysis to release target proteins, which has been successfully applied to large-scale recovery of products such as interleukin-2. Interestingly, certain recombinant antibodies can spontaneously leak into the culture medium without active lysis treatment. Their distribution ratio is regulated by promoter strength, induction conditions and specific amino acid mutations. This passive secretion phenomenon provides new insights for the development of continuous fermentation processes, enabling gradient release of periplasmic proteins without impairing cell viability by finely regulating culture parameters, thus balancing product yield and biological activity.

It was conventionally acknowledged that the reducing cytoplasmic environment of E. coli impedes disulfide bond formation. Nevertheless, genetically engineered oxidative cytoplasm can be constructed. Commercially available strains including AD494, Origami and the novel Shuffle strain have realized the correct intracellular folding of complex proteins such as collagenase and antibody receptor domains. These systems break new ground for recombinant protein production by expanding the expression scope of disulfide bond-dependent proteins from the periplasm to the cytoplasm.

Chaperone proteins and isomerases synergistically increase recombinant protein yields by suppressing non-functional aggregation of folding intermediates. In oxidative cytoplasmic strains, co-expression of DsbC isomerases markedly improves the correct folding of disulfide bond-dependent proteins such as scFv and variable heavy-chain domain antibodies (VHH), whose mechanism involves catalyzing the rearrangement of non-native disulfide bonds.

The thioredoxin fusion strategy presents dual superiorities. In wild-type strains, it stabilizes disulfide bond-free eukaryotic proteins via chaperone effects; in oxidative cytoplasm, it directly participates in disulfide bond formation, enabling the functional expression of proteins with multiple disulfide bonds such as LEKTI domains and c-Met receptor scFv. Furthermore, thioredoxin fusion can reverse the aggregation tendency of complex proteins like BSPH1 in mutant strains and generate soluble bioactive products. These strategies systematically optimize the intracellular folding pathway of complex proteins by coupling oxidative microenvironments with folding auxiliary factors.

Among oxidative cytoplasmic strains, double mutants are more conducive to the functional expression of disulfide bond-dependent proteins than single mutants. Combined application of double mutations and DsbC isomerases significantly elevates the yields of scFv and VHH antibodies through mediating disulfide bond rearrangement and stabilizing folding intermediates.

In vitro renaturation serves as an essential remedial strategy for proteins prone to irreversible aggregation. Alkaline buffers combined with redox pairs facilitate disulfide bond formation; low temperature and dynamic oxidation gradients restrain erroneous protein aggregation. Immobilization technology reduces intermolecular interactions among folding polypeptides, and supplementation of chaperones or isomerases rectifies misfolding defects. The above approaches systematically optimize the in vitro folding process of complex proteins.

Moreover, cytoplasmic expression is also influenced by vectors, protein engineering strategies and type I extracellular secretion pathways. Firstly, fusion chaperones effectively enhance the folding efficiency of recombinant proteins in the E. coli cytoplasm. Beyond thioredoxin, C-terminal maltose-binding protein fusion stabilizes specific scFv fragments, while NusA fusion is indispensable for the active expression of certain scFv and APRIL proteins. Although SUMO tags improve the solubility of partial scFv, systematic evaluation of their impacts on the structural quality of disulfide bond-containing proteins remains insufficient.

To eliminate repetitive experimental trials, protein engineering has been adopted to design stable disulfide bond-independent mutants, yielding functional scFv and VHH antibodies capable of folding in reducing cytoplasm, which lays a foundation for intracellular immunological applications. In addition, innovative application of the Gram-negative bacterial type I secretion pathway achieves efficient secretion of antibody fragments. Fusion of scFv or VHH with the C-terminal domain of α-hemolysin enables direct secretion into culture media without periplasmic signal peptides while retaining oxidative biological activity, establishing an efficient production platform for vaccine development and in vitro diagnostics.

The optimization of protein production has evolved from traditional trial-and-error methods to rational design based on folding mechanisms. Despite diverse established expression tools, uncertainties still exist regarding the functional expression of disulfide bond-dependent proteins in bacteria. Systematic optimization procedures should be implemented stepwise: prioritize the optimization of DNA sequences and expression conditions, select appropriate subcellular localization pathways for target proteins, and match corresponding signal peptides and host strains accordingly.

This paper systematically summarizes multiple strategies to promote the recombinant expression of disulfide bond-dependent proteins in E. coli from the perspectives of periplasmic and cytoplasmic expression, aiming to provide practical references for relevant scientific research and industrial applications.