Insight

Recombinant protein expression aims to produce proteins with desirable solubility, folded conformation and biological activity using appropriate host cells. Environmental composition and optimization serve as core determinants of soluble recombinant protein expression. Protein folding is a biophysical process. Upon induced protein synthesis, misfolding, proteolysis and aggregation frequently occur. Failure to fold into native three-dimensional structures generally yields inactive proteins, and may also trigger functional alternation or cytotoxicity. Such defects can be mitigated via optimized production and folding parameters.

Moreover, recombinant protein expression and post-translational modification facilitate exploration of gene biological functions, as well as biochemical, enzymatic and structural biology research. Folding condition optimization is pivotal in recombinant protein manufacturing. Parameters including expression vectors, culture medium composition, growth temperature and molecular chaperones can be modulated to markedly enhance folding efficiency and product yield. Low cultivation temperature slows down protein synthesis and allows sufficient time for proper folding, yet it may concurrently reduce production output. Therefore, balanced parameters must be established to maintain yield while achieving optimal folding performance.

Protein folding remains a predominant bottleneck in recombinant protein manufacturing. Despite theoretical simplicity of protein acquisition, practical production commonly encounters inclusion body formation, protein aggregation, denaturation, misfolding, aberrant post-translational modification and even suppressed protein expression. Root cause analysis is prerequisite for troubleshooting, with genome sequencing acting as the fundamental approach for protein structural characterization.

Folding optimization is predominantly realized by adjusting ambient parameters such as temperature and inducer concentration. Reduced temperature decelerates protein biosynthesis and favors correct folding. High-dose molecular chaperones also facilitate folding progression. Nevertheless, highly unstable proteins may still undergo misfolding even with chaperone assistance, and single chaperone species often fail to exert satisfactory effects, necessitating multi-factor collaborative regulation. Protein refolding refers to the conformational transition from denatured state to native structure, which is closely correlated with denaturant concentration.

Hybrid protein technology emerges as an alternative strategy to alleviate folding issues by elevating target protein solubility. Nonetheless, this technique still has limitations, including soluble aggregate generation, impaired bioactivity and unpredictable solubility improvement effects.

Molecular chaperones exert protective effects throughout protein folding processes to avert misfolding, and participate in protein folding, refolding, quality control and structural stabilization, preventing native protein denaturation and abnormal accumulation. Rational chaperone selection effectively boosts recombinant protein stability and correct folding. Possessing adenosine triphosphatase (ATPase) activity, chaperones execute physiological functions in a substrate-specific manner.

Based on functional mechanisms and substrate interaction patterns, molecular chaperones are categorized into three groups: folding chaperones, holding chaperones and disaggregating chaperones. Folding chaperones mediate ATP-dependent substrate conformational rearrangement, diminish aggregate formation and degrade misfolded polypeptides, thereby inhibiting inclusion body generation.

Low recombinant gene expression levels are mainly attributed to inferior mRNA stability, secondary structure formation at 5′-terminal mRNA, rare codon usage and weak Shine-Dalgarno sequences. Cells eliminate misfolded proteins as a self-defense response. The endogenous protein quality control system protects cells from defective translation products and damaged polypeptides. It binds aberrant peptide chains to regulate folding dynamics and restrain aggregation, eliminating misfolded monomers and defective proteins. Excessive accumulation of misfolded proteins may overwhelm intracellular degradation systems and eventually induce cellular damage.

Post-translational modification constitutes the final stage of protein biosynthesis, encompassing reversible and irreversible chemical alterations of translated polypeptides. These modifications profoundly determine protein spatial conformation, physiological functions and biological activity. Common modification types include phosphorylation, glycosylation, acetylation, ubiquitination, SUMOylation and methylation.

Glycosylation represents the most prevalent and sophisticated modification in eukaryotic cells, critically influencing protein folding, stability, solubility and bioactivity. Glycoproteins occupy a vital position in therapeutic protein development, and glycosylation modification enhances their structural stability and therapeutic efficacy.

Acetylation modulates extensive cellular processes such as chromatin stability, cell cycle progression, metabolism, transcription, gene expression and protein homeostasis, occurring in the majority of eukaryotic proteins. Phosphorylation is a reversible modification that introduces phosphate groups to cytoplasmic and nuclear proteins, regulating cell signaling, differentiation, DNA repair, stress response and apoptosis. Methylation was initially studied in histones, and lysine methylation of non-histone proteins has also been verified as an essential regulatory mechanism. S-nitrosylation achieves covalent bonding between nitric oxide and cysteine thiol groups, modulating enzymatic activity, protein interaction and signal transduction cascades.

Multiple computational and experimental approaches are applied to characterize post-translational modifications and modification sites. Computational identification relies on bioinformatic analysis of experimental datasets. Dedicated experimental methods are adopted for different modification types; in vitro ubiquitination assays are utilized to investigate protein degradation mechanisms, while polymerase chain reaction (PCR) enables quantitative detection of site-specific modifications.

High-performance liquid chromatography coupled with mass spectrometry serves as the mainstream platform for glycosylation profiling and glycan structure analysis. Glycan chains are released via enzymatic or chemical cleavage, followed by liquid chromatography-mass spectrometry (LC-MS) characterization. Fluorescent labeling and electrophoretic separation, combined with glycan-specific lectins and antibodies, further improve detection sensitivity of glycosylated proteins.

Recombinant protein expression underpins modern biotechnology. Multiple obstacles including proteolysis, misfolding, inclusion body formation and aggregation severely compromise protein structure and functional integrity. Targeted strategies have been developed, such as parameter optimization, synthesis duration regulation, chaperone application and folding buffer formulation, together with robust intracellular quality control systems.

Post-translational modification is indispensable for recombinant protein production and clinical application. Clarification of its biological significance and maturation of identification techniques facilitate the establishment of high-efficiency production protocols, accelerate drug and therapeutic regimen development, and provide novel remedies for malignant tumors, neurological disorders and various acute and chronic diseases.