1. Definition of Inclusion Bodies
Inclusion bodies (IBs) are high-density, insoluble protein granules encapsulated by membranes, formed during high-level heterologous gene expression in prokaryotic cells, especially Escherichia coli. They appear as high-refractive regions under microscopic observation and are distinctly differentiated from other cytoplasmic components. IBs exist as either membrane-wrapped aggregates or bare amorphous structures, featuring water insolubility.
Typically composed of over 50% recombinant proteins, the residual components include ribosomal elements, RNA polymerase, endotoxin, outer membrane proteins OmpC, OmpF, OmpA, circular/nicked plasmid DNA, liposomes and lipopolysaccharides. With a particle size of 0.5-1 μm and a density of approximately 1.3 mg/mL, IBs are amorphous and non-water-soluble, only soluble in denaturants such as urea and guanidine hydrochloride.
The formation mechanism of inclusion bodies is sophisticated. Excessively high nascent polypeptide concentration leaves insufficient time for proper protein folding, resulting in amorphous protein aggregation. Cultivation parameters including medium composition, temperature, pH value and ionic strength also exert significant impacts. Functional proteins maintain specific three-dimensional conformations, whereas proteins trapped in inclusion bodies stay unfolded and biologically inactive.
2. Impacts of Inclusion Bodies on Fermentation Products
Advantages
High density facilitates separation and purification procedures.
Protect recombinant proteins from degradation by endogenous proteases in E. coli.
Ideal expression form for proteins with cytotoxicity under native conformation.
Disadvantages
Proteins within inclusion bodies adopt misfolded conformations and lose biological activity, limiting product application, structural analysis and proteomics research. In most cases, insoluble inclusion bodies require renaturation to recover soluble functional proteins.
3. Formation Mechanism of Inclusion Bodies
Excessive expression level: Rapid peptide synthesis outpaces folding rate, triggering disulfide bond mismatching and non-specific intermolecular binding. Aggregation dominates over correct folding under high expression intensity.
Deficient glycosylation: Absence of glycosylation modification for eukaryotic heterologous proteins reduces intermediate solubility and induces aggregation.
Folding obstruction: Secretory signal sequences interfere with normal protein folding and generate misfolded molecules.
Amino acid composition: Proteins rich in sulfur-containing amino acids tend to form inclusion bodies more easily.
Intermediate aggregation: Partially denatured protein intermediates polymerize into inclusion bodies. pH near isoelectric point, improper ionic strength and temperature accelerate aggregation.
Intermolecular chemical bonds: Ionic bonds, hydrophobic interactions and covalent bonds drive protein aggregation during bacterial secretion.
Suboptimal cultivation conditions: Inferior culture environment inhibits active protein expression and promotes inclusion body generation.
Insufficient folding accessories: Deficiency of foldases and molecular chaperones restrains correct polypeptide folding.
4. Control Strategies for Inclusion Bodies
4.1 Prevention of Inclusion Body Formation
4.1.1 Co-expression with Molecular Chaperones
Molecular chaperones assist polypeptide assembly and dissociate from mature functional proteins without constituting structural components. Co-expression effectively alleviates misfolding and elevates soluble protein yield.
Common chaperone systems in E. coli consist of Hsp60 family (GroEL, GroES) and Hsp70 family (DnaK, GrpE, DnaJ). GroEL mediates protein transportation between soluble and insoluble fractions, while DnaK suppresses protein aggregation. Synergistic combinations of multiple chaperones markedly improve folding efficiency. Matching chaperone types with protein properties, promoters, signal peptides and low-temperature cultivation further optimize soluble expression.
4.1.2 Genetic Modification
Amino acid sequence determines protein folding tendency. Site-directed mutagenesis alters hydrophobicity and structural stability to inhibit aggregation without impairing bioactivity. Computer-aided homologous modeling improves mutation design accuracy.
Random mutagenesis technologies including error-prone PCR, chemical mutagenesis and DNA shuffling are widely applied in directed protein evolution for solubility enhancement.
4.1.3 Fusion Expression
Soluble fusion tags assist folding and reduce aggregation. Classic tags include MBP, GST, NusA, DsbA and TrxA. MBP and NusA exhibit outstanding solubility-promoting capacity, while DsbA performs well for disulfide bond-containing proteins.
Five mainstream mechanisms explain tag functions:
Electrostatic shielding: Highly charged tags generate intermolecular repulsion to avoid aggregation.
Micelle model: Hydrophilic tags encapsulate hydrophobic protein domains to maintain solubility.
Entropy anchor model: Tags restrict polypeptide movement and reserve sufficient time for folding.
Chaperone magnet model: Specific tag sequences recruit endogenous chaperones to assist folding.
Chaperone mimicry: Fusion tags act as chaperones to interact with target proteins directly.
4.1.4 Medium Optimization
pH value should be kept far from the protein isoelectric point to minimize aggregation. Additives including sorbitol, betaine, Triton X-100 and glycine enhance cytoplasmic osmotic protection and cell membrane permeability, drastically increasing soluble protein production.
4.1.5 Reduction of Protein Synthesis Rate
Low cultivation temperature slows down bacterial metabolism and peptide synthesis, allowing adequate folding time and reducing protein degradation. The feasible temperature range for E. coli growth is 10–37 °C.
Induction conditions are strictly optimized. Low-concentration inducer, appropriate induction timing and cell density effectively balance expression level and solubility. Fed-batch cultivation and sufficient aeration support high-density induction of soluble proteins.
4.1.6 Selection of Optimal Expression Systems
Strains favorable for disulfide bond formation such as Rosetta Origami(DE3), Origami(DE3) and Origami B(DE3) are preferred for corresponding proteins. Host selection depends on protein molecular weight, disulfide bond quantity, localization and modification requirements.
Codon optimization eliminates codon usage bias when expressing eukaryotic proteins in E. coli. Prokaryotic proteins are conventionally expressed in native prokaryotic hosts due to unnecessary eukaryotic modification functions.
4.2 Renaturation Procedures of Inclusion Bodies
4.2.1 Harvest and Lysis of Bacterial Cells
Cool fermentation broth first. Centrifugation and filtration separate intact cells and inclusion bodies from crude lysate. Inclusion bodies withstand strong shear force, enabling diverse cell lysis methods.
4.2.2 Washing of Inclusion Bodies
Low-concentration denaturant and mild detergents remove membrane fragments, exogenous DNA and miscellaneous proteins. Standard washing buffer formula: 20 mmol/L Tris-HCl (pH 8.0), 0.5 mol/L NaCl, 2 mol/L urea, 2% Triton X-100. Conduct stirring washing for 20–30 min, followed by centrifugation at 12,000 rpm and 4 °C for 15 min. Repeat 2–3 cycles, then rinse with Tris-HCl solution to eliminate residual impurities.
4.2.3 Solubilization of Inclusion Bodies
High-concentration urea or guanidine hydrochloride breaks intermolecular bonds and unfolds polypeptide chains. Guanidine hydrochloride serves as a stronger denaturant. Reducing agents such as DTT and β-mercaptoethanol eliminate abnormal disulfide bonds in cysteine-rich proteins.
Solubilization buffer formulation: 20 mmol/L Tris-HCl (pH 8.0), 0.5 mol/L NaCl, 8 mol/L urea, 0.2 mmol/L DTT, 2% Triton X-100. Suspend precipitates and stir overnight at room temperature, then collect supernatant after centrifugation.
4.2.4 Protein Renaturation
Gradual removal of denaturants facilitates spontaneous refolding into native conformation and correct disulfide bond rearrangement. Common renaturation approaches include dialysis renaturation, on-column renaturation and dilution renaturation. Effective urea concentration range for renaturation: 2 M–4 M; guanidine hydrochloride range: 1.5 M–4 M.
4.2.5 Result Evaluation
Bioactivity assay, RP-HPLC, spectrophotometry, ligand binding test, ELISA and biological characterization are adopted to verify native protein conformation and calculate renaturation recovery rate.
