1. Basic Overview of Inclusion Bodies
1.1 Definition of Inclusion Bodies
Inclusion bodies (IBs) are intracellular macromolecular aggregates predominantly composed of proteins, distributed in the nucleus, cytoplasm or periplasmic space. Their formation stems from the imbalance among protein folding, aggregation and degradation processes, triggered by exogenous or mutated gene expression, as well as cellular environmental stress that hinders proper protein folding and post-translational modification. Mostly presented as dense insoluble protein granules with disordered amorphous structures, IBs exist either membrane-wrapped or naked. Their morphology and properties vary depending on formation causes and host cell types.
1.2 Formation Mechanism of Inclusion Bodies in Escherichia coli
IB formation in E. coli is a sophisticated multi-protein involved process affected by diverse factors. Proteins with high expression levels, large molecular weight, abundant hydrophobic residues and disordered structures are prone to aggregate into IBs. Under overexpression conditions, protein synthesis rate far exceeds the time required for correct folding, raising misfolding probability. Increased energy consumption induces cellular stress and metabolic burden, further exacerbating protein misfolding.
As prokaryotes, E. coli lack subcellular structures essential for complete post-translational modifications including glycosylation and disulfide bond formation. Disulfide bond establishment serves as the rate-limiting step for proper folding of disulfide-rich proteins, and the limited disulfide bond formation capacity of E. coli leads to high tendency of misfolding and aggregation. Additionally, cultivation temperature, pH value and other ambient conditions also exert impacts on IB accumulation during recombinant protein production.
1.3 Characteristics of Protein Inclusion Bodies in E. coli
Most bacterial IBs exist as amorphous aggregates devoid of natural structure and biological functions. Nevertheless, amyloid-like ordered structures and corresponding bioactivity have been discovered in IBs of E. coli-expressed β-galactosidase and asparaginase.
IBs are primarily homogeneous self-aggregates of target proteins, regarded as a vital form of high-purity recombinant proteins with potential development and utilization value.
IBs specifically localize at cell poles or division sites in E. coli, resulting in asymmetrical inheritance that only one daughter cell retains IBs after cell division.
Lysate of IB-containing E. coli presents milky turbid liquid state due to light scattering effect of aggregated particles.
2. Strategies to Mitigate Inclusion Body Formation
2.1 Optimization of Bacterial Culture Conditions
Modulating cultivation parameters effectively elevates soluble protein yield and suppresses IB generation. Key adjustable factors include growth temperature, inducer dosage, medium additives and stable pH maintenance, among which temperature and inducer concentration regulation are the most widely adopted approaches.
Low-temperature cultivation is a practical optimization method. A two-stage cultivation protocol is commonly applied: cells are first cultured rapidly at 37 °C or treated with short-term heat shock at 47 °C for 30 minutes, then the temperature is lowered to 15–20 °C to initiate mild target protein expression. This protocol facilitates correct folding and soluble expression of multiple proteins such as Tri101 acetyltransferase, DepA and DepB.
Controlled IPTG concentration and glucose supplementation efficiently slow down protein synthesis rate, relieve metabolic pressure and create favorable conditions for native protein folding. Isopropyl β-D-1-thiogalactopyranoside (IPTG) acts as the chemical inducer of lactose operon to activate target gene transcription and expression.
2.2 Host Engineering for Soluble Recombinant Protein Expression
2.2.1 Enhancement of Disulfide Bond Formation
Engineered strains including Origami and Shuffle establish intracellular oxidative microenvironment conducive to disulfide bond formation. The CyDisCo system also remodels cellular redox homeostasis and remarkably improves protein solubility.
2.2.2 Strain Modification for Improved N-Glycosylation
Abnormal glycosylation causes protein aggregation and IB accumulation. Introduction of key glycosylation enzymes such as PglB promotes glycosylation reaction, enhancing protein folding efficiency and structural stability. Leaky glycosylation strains constructed via lpp gene knockout or mutation improve outer membrane permeability, supporting recombinant expression of glycoproteins including glycosylated antibody fragments.
2.2.3 Chaperones and Foldases for Misfolding Correction
Protein misfolding constitutes the primary cause of IB formation. Foldases such as protein disulfide isomerase (PDI) and peptidyl-prolyl cis-trans isomerase (PPI) assist polypeptide chains in conformational rearrangement. Molecular chaperones stabilize unfolded intermediates, facilitate protein assembly and inhibit aggregate formation.
2.2.4 Expression System Optimization for Membrane and Toxic Proteins
Beneficial gene mutations reduce transcriptional activity and restrain IB generation. Strains C41(DE3), C43(DE3) and BL21(DE3) pLysS downregulate T7 RNA polymerase expression and alleviate cytotoxicity induced by excessive membrane protein expression. The inhibitor Gp2 of T7 phage RNA polymerase applied in BL21(DE3):TN7 decouples recombinant protein synthesis from host cell growth, effectively reducing IB accumulation.
2.2.5 Engineering of Metal Ion Transport and Metalloenzyme Metabolic Pathways
Metal cofactors are indispensable for folding and functional activation of metalloenzymes. Overexpression of operons related to metal ion uptake and transport pathways also alleviates protein aggregation.
2.3 Construction of Novel Expression Vectors
Vector modification is an effective means to enhance protein solubility and inhibit IB formation. Fusion expression with soluble polypeptides or chaperone proteins optimizes folding behavior and avoids aggregation. Weak promoters and low-copy plasmids moderate protein synthesis velocity. Dual-plasmid co-expression strategies integrate advantages of high and low copy vectors, enabling efficient production of soluble and bioactive protein complexes.
2.4 Modification of Target Protein Sequence
Bioinformatics platforms including Protein-Sol and SoDoPE identify aggregation-prone amino acid sequences, and site-directed mutagenesis can reduce protein self-assembly tendency. Target proteins can be transported to periplasmic space guided by signal peptides such as OmpA and PhoA. The oxidative periplasmic environment and endogenous foldases promote disulfide bond maturation, acquiring proteins with improved solubility and biological activity.
3. Potential Industrial Application Advantages of Inclusion Bodies
Although IB-derived proteins lose native bioactivity and require denaturation and refolding to recover spatial conformation and functions, IBs possess prominent industrial application prospects. IBs feature excellent mechanical and chemical stability, serving as promising biomaterials for drug delivery and protein encapsulation. Their water-insoluble and dense aggregated properties simplify separation and purification procedures, suitable for large-scale industrial manufacturing.
Beyond recombinant protein production, IBs can act as critical precursors for pharmaceutical synthesis and efficient expression carriers of commercially valuable antimicrobial peptides. Practical industrial applications have achieved fruitful outcomes; for instance, IB-based biosynthesis realizes higher 1-butanol yield than conventional techniques, laying foundation for its expansion in cosmetic and food industries.
Conclusion
Comprehensive solutions for restraining IB formation cover expression rate regulation, host strain engineering for complete post-translational modification, vector optimization and bioinformatics-assisted protein sequence redesign. Further exploration of IB formation mechanism and renaturation technology will drive innovative application breakthroughs. Interdisciplinary research combining biology, chemistry and engineering will develop high-efficiency and stable IB production platforms, expand application boundaries and accelerate industrial commercialization.
