In recent years, recombinant proteins have found expanding applications in industry, agriculture and biomedicine. Featuring high expression yields, short cultivation cycles and low production costs, Escherichia coli remains one of the most prevalent bacterial hosts for industrial manufacturing.
Nevertheless, a major technical bottleneck frequently emerges when expressing recombinant proteins in E. coli: target proteins tend to accumulate as insoluble, biologically inactive aggregates known as inclusion bodies (IBs). This poses substantial challenges for recovering soluble proteins with native folding and biological activity at both laboratory and industrial scales. Through in-depth investigations into the formation mechanisms, influencing factors and intrinsic properties of IBs, academia and industry have developed a suite of effective approaches to mitigate inclusion body formation in E. coli.
1. Inclusion Bodies: Definition and Characteristics
1.1 Definition of Inclusion Bodies
Inclusion bodies (IBs) are macromolecular proteinaceous aggregates formed intracellularly, which can reside in the nucleus, cytoplasm or periplasmic space. Their emergence generally arises from an imbalance among protein folding, assembly and degradation, triggered by heterologous gene overexpression, accumulation of mutated proteins, or folding machinery overload under cellular stress. Morphologically, IBs are dense, insoluble protein particles with mostly amorphous structures; some are membrane-bound, whereas others exist as naked aggregates.
1.2 Mechanisms Underlying Inclusion Body Formation
The formation of inclusion bodies in E. coli represents a complex process governed by multiple interacting factors:
Intrinsic protein properties: Proteins with high expression levels, large molecular weights, abundant hydrophobic residues and intrinsically disordered regions are more prone to aggregation into IBs.
Dysregulated expression kinetics: Under strong induction conditions, the rate of protein synthesis outpaces the rate of correct folding, drastically elevating the probability of misfolding. Concurrently, elevated energy demand imposes metabolic burden and cellular stress, further exacerbating misfolding.
Insufficient capacity for post-translational modification: As a prokaryote, E. coli lacks subcellular machineries required for complex post-translational modifications such as glycosylation. Particularly for disulfide bond-containing proteins, limited cytoplasmic capacity for disulfide bond formation becomes a rate-limiting step for proper folding, readily leading to misfolding and aggregation.
Environmental conditions: Fluctuations in cultivation parameters including temperature and pH markedly affect the propensity for inclusion body formation.
1.3 Distinctive Properties of Inclusion Bodies
Structural features: IBs are predominantly amorphous aggregates lacking native conformation and biological function. Nevertheless, ordered amyloid-like structures with partial functionality have been documented in certain systems such as β-galactosidase and asparaginase.
High homogeneity: E. coli-derived IBs consist largely of homogeneous self-assembled target proteins, which renders them a high-purity form of recombinant protein with exploitable potential.
Polar localization: IBs preferentially accumulate at cellular poles or division sites. Such asymmetric distribution results in only one daughter cell inheriting IBs upon cell division.
Light scattering effect: After cell lysis, suspensions containing IBs exhibit prominent turbidity resembling milky homogenate due to light scattering.
2. Five Major Strategies to Alleviate Inclusion Body Formation
2.1 Optimization of Cultivation Conditions
Modulating culture parameters represents a straightforward and readily implementable strategy. Key variables include temperature, inducer concentration, medium additives and pH.
Low-temperature induction: A two-stage cultivation scheme is commonly adopted: biomass accumulation at 37 °C followed by induction at 15–20 °C. This approach effectively improves the solubility of numerous proteins including Tri101 acetyltransferase and DepA/DepB. Short-term heat pre-shock (47 °C, 30 min) serves as an alternative viable tactic.
Precise inducer tuning: IPTG acts as the canonical inducer for the lactose operon. Reducing IPTG dosage or supplementing media with glucose suppresses transcriptional strength of the target gene, alleviates metabolic stress and allows sufficient time for nascent polypeptides to fold correctly.
2.2 Engineering of Host Strains
Host engineering remodels the intracellular environment to favor proper folding, constituting a fundamental approach to reduce IB generation.
Facilitating disulfide bond formation: Engineered strains such as Origami and SHuffle possess oxidizing cytoplasm that supports accurate disulfide pairing. The CyDisCo system achieves comparable improvements in soluble expression of disulfide-rich proteins via redox homeostasis regulation.
Enhancing N-glycosylation capacity: Aberrant glycosylation promotes protein aggregation. Reconstruction of the N-glycosylation pathway via introduction of key enzymes (e.g., PglB) or utilization of lpp mutant strains with improved outer membrane permeability facilitates correct folding and secretion of glycoproteins including antibody fragments.
Co-expression of folding assistants: Covalent co-expression of foldases (PDI, PPI) or molecular chaperones stabilizes unfolded intermediates, directs them toward native folding pathways and suppresses aggregation.
Host adaptation for toxic proteins: For cytotoxic proteins such as membrane proteins, mutant strains including C41(DE3), C43(DE3) and BL21(DE3)pLysS are widely applied. These strains feature attenuated T7 RNA polymerase activity to moderate transcription intensity and mitigate host toxicity. BL21(DE3):TN7 utilizes Gp2, an inhibitor of T7 phage RNA polymerase, to decouple protein expression from host growth and lower IB yields.
Augmenting supply of metal cofactors: Adequate cofactor availability is a prerequisite for correct folding of metalloenzymes. Overexpression of operons governing metal ion uptake elevates intracellular cofactor accessibility and reduces aggregation stemming from cofactor deprivation.
2.3 Rational Design of Expression Vectors
Vector architecture directly governs transcriptional intensity and translational velocity, serving as a critical control point for IB formation.
Fusion tag technology: Fusion of the target protein with highly soluble tags (MBP, GST), molecular chaperones or folding-enhancing peptides improves solubility and guides proper folding (Figure 3).
Weak promoters and low-copy plasmids: Employment of weak promoters or low-copy-number vectors slows down protein synthesis, enabling the endogenous folding machinery to process nascent chains efficiently. Dual-vector co-expression systems combining high-copy and low-copy plasmids demonstrate promising performance for soluble production of multi-subunit protein complexes.
2.4 Engineering of Target Protein Sequence
The amino acid sequence intrinsically determines protein folding behavior; engineering the primary sequence mitigates aggregation propensity at the source.
Bioinformatic prediction and site-directed mutagenesis: Tools such as Protein-Sol and SoDoPE identify aggregation-prone hotspots within protein sequences. Site-directed mutagenesis eliminates these unfavorable features to enhance solubility.
Redirection of subcellular localization: Target proteins can be directed to the periplasm via signal peptides (OmpA, PhoA). The periplasm provides an oxidizing microenvironment enriched with folding enzymes that promote disulfide bond formation, yielding soluble proteins with biological activity.
3. Inclusion Bodies Are Not Intrinsically Disadvantageous — Unique Industrial Value
Although proteins within IBs generally lose native conformation and bioactivity and require denaturation and refolding to recover functionality, their distinctive physicochemical properties confer advantages in certain industrial scenarios:
Superior mechanical and chemical stability: IBs maintain structural integrity under extreme pH, temperature and protease exposure, making them promising building blocks for biomedical applications such as drug delivery vehicles and protein encapsulation materials.
Simplified separation and purification: Owing to high density and low aqueous solubility, IBs can be separated from most soluble host contaminants via simple centrifugation, streamlining downstream purification workflows especially for large-scale manufacturing.
Potential for functional exploitation: IBs can act as stable storage forms of recombinant proteins and serve as expression and purification platforms for pharmaceutical precursors and antimicrobial peptides.
Practical production examples: In biosynthesis of bulk chemicals such as 1-butanol, IBs utilized as immobilized enzyme carriers achieve higher productivity than conventional routes, indicating broad applicability in cosmetics, food and related industries.
4. Conclusions and Outlook
Reducing inclusion body formation in E. coli represents a systematic engineering challenge spanning multiple layers. Current strategies encompass cultivation optimization, host strain engineering, vector design and protein sequence modification, delivering substantial improvements. Future advances in understanding IB nucleation, folding regulatory networks and refolding technologies will enable the development of smarter, more efficient production platforms. Interdisciplinary integration of synthetic biology, structural biology and process engineering will further expand the scope of IB applications and drive recombinant protein manufacturing toward higher efficiency and broader utilization。
