Insight

Molecular chaperones are a class of proteins widely present in prokaryotes and eukaryotes. Their core functions include assisting protein correct folding, assembly, transportation and degradation, as well as mediating the proper assembly of other proteins, without becoming components of the final functional structure. With continuous in-depth research, an increasing number of molecular chaperones have been identified. This article mainly introduces cytoplasmic chaperones localized in the cytoplasm and periplasmic chaperones distributed in the periplasmic space of Gram-negative bacteria.

Cytoplasmic molecular chaperones encompass diverse subtypes, mainly including heat shock proteins (Hsps), prefoldin, trigger factor (TF), etc. Among them, heat shock proteins constitute the largest family of molecular chaperones. Classified by molecular weight, they are divided into small heat shock proteins (sHsp), Hsp40, Hsp60, Hsp70, Hsp90 and Hsp100 families.

Heat shock proteins possess two typical characteristics: firstly, their expression can be induced under stress conditions to help cells and organisms resist adverse environments; secondly, they are highly evolutionarily conserved, with highly similar amino acid sequences and three-dimensional structures across prokaryotes, eukaryotes and various species.

Periplasmic molecular chaperones reside in the periplasmic space of Gram-negative bacteria. As an oxidative microenvironment, the periplasmic space facilitates disulfide bond formation, and numerous secretory proteins containing disulfide bonds complete their folding process here. Periplasmic molecular chaperones are categorized into four groups: general molecular chaperones, specific molecular chaperones, peptidylprolyl isomerases (PPIases), and proteins involved in disulfide bond formation.

Escherichia coli is one of the most commonly used hosts for recombinant protein production. However, its application is frequently restricted by intracellular protein aggregation into inclusion bodies and misfolding of newly synthesized nascent polypeptides during translation, which leads to the loss of natural protein conformation and biological functions. Co-expression of molecular chaperones serves as an effective strategy to solve these problems. Acting as protein folding regulators, molecular chaperones assist newly synthesized, aggregated or misfolded proteins to refold into native conformations, and have been widely applied to enhance the expression level of hard-to-produce recombinant proteins in E. coli.

Trigger factor possesses peptidylprolyl isomerase activity, and is the sole ATP-independent ribosome-associated molecular chaperone in E. coli. It binds to the L23 protein of the large ribosomal subunit to form a cradle-like structure, which shields nascent polypeptide chains from misfolding and intracellular degradation.

Trigger factor consists of three distinct domains: the N-terminal domain responsible for ribosome binding, the middle domain with PPIase catalytic activity, and the C-terminal domain for substrate protein recognition and binding. It interacts with hydrophobic regions of target proteins via internal hydrophobic residues to effectively inhibit protein aggregation.

For instance, co-expression of trigger factor with canine parvovirus VP2 protein can remarkably improve the solubility and immunogenicity of VP2 protein, which supports large-scale production of virus-like particles. Moreover, trigger factor functions synergistically with classic chaperone systems including GroEL/GroES and DnaK/DnaJ/GrpE to further elevate the soluble expression yield of recombinant proteins.

Belonging to the Hsp60 family, the GroEL/GroES complex is the predominant folding-assisted molecular chaperone system in E. coli. GroEL is an oligomeric barrel-shaped protein assembled by 14 subunits, forming two stacked heptameric rings with a central cavity capable of encapsulating unfolded polypeptides. As the co-chaperone of GroEL, GroEL binds to GroEL to form a confined mini-cage, constructing a hydrophilic microenvironment favorable for protein folding.

The functional cycle proceeds as follows: GroEL first captures unfolded target proteins, followed by GroES docking to seal the folding chamber. Driven by ATP hydrolysis, GroES and ADP dissociate from GroEL, and correctly folded proteins are released. Polypeptides failing to achieve native conformation will re-enter the GroEL/GroES system for iterative refolding.

Practical applications have verified that co-expression of GroEL/GroES with human papillomavirus L1 protein significantly boosts the soluble expression of L1 protein, laying a solid foundation for HPV vaccine development and production. This system also exhibits synergistic folding effects with the DnaK/DnaJ/GrpE system to further optimize recombinant protein solubility.

DnaK is the prokaryotic homolog of eukaryotic Hsp70 and an ATP-dependent molecular chaperone. It binds to unfolded or partially folded proteins to prevent intermolecular aggregation and mediate orderly correct folding. The biological functions of DnaK rely on two auxiliary co-chaperones: DnaJ and nucleotide exchange factor GrpE.

DnaJ interacts with DnaK to accelerate ATP hydrolysis and strengthen the binding affinity between DnaK and substrate proteins; GrpE facilitates ADP dissociation from DnaK, enabling DnaK to rebind ATP and ultimately release folded target proteins.

In biopharmaceutical research, co-expression of the DnaK/DnaJ/GrpE system with anti-TNF-α Fab antibody effectively enhances the solubility and biological activity of antibody fragments. Combined application with the GroEL/GroES system can achieve more prominent improvement in recombinant protein soluble expression.

ClpB is classified into the Hsp100 chaperone family and exhibits potent disaggregase activity. It utilizes energy released from ATP hydrolysis to disassemble dense protein aggregates into individual monomeric polypeptide chains.

The disaggregation function of ClpB is strictly dependent on the DnaK/DnaJ/GrpE system. DnaK mediates the recruitment of ClpB to aggregated protein substrates and initiates the disaggregation reaction. In recombinant protein manufacturing, ClpB is usually co-expressed with the DnaK/DnaJ/GrpE and GroEL/GroES cascades to synergistically eliminate protein aggregation and increase soluble protein output.

Yeast is a mature eukaryotic expression system for heterologous protein production. Heterologous protein folding and assembly can be efficiently promoted via endogenous or exogenous molecular chaperones, thus improving the overall expression quality and production yield.

The transport process of secretory proteins from the endoplasmic reticulum (ER) to the Golgi apparatus is the major rate-limiting step in the yeast secretory pathway. The endoplasmic reticulum is the core site for intracellular disulfide bond formation and protein folding, where multiple molecular chaperones participate in the whole folding process. Nascent polypeptides anchored on the endoplasmic reticulum can maintain soluble status through specific interactions with ER-resident chaperones. The main ER-localized molecular chaperones include protein disulfide isomerase (PDI), binding immunoglobulin protein (BiP) and calreticulin. Numerous studies have confirmed that co-expression of molecular chaperones with target genes can markedly upregulate heterologous protein expression levels.

As a core member of the protein disulfide isomerase family, PDI catalyzes the formation, cleavage and rearrangement of intracellular disulfide bonds, ensuring accurate oxidative folding of secretory proteins such as monoclonal antibodies and polypeptide hormones. It works synergistically with endoplasmic reticulum oxidoreductin 1 (ERO1) to stabilize the oxidative redox environment inside the endoplasmic reticulum.

Experimental data show that co-expression of PDI alone and combined ERO1-PDI in Pichia pastoris increases the enzymatic activity of glucose oxidase by 29.7% and 100% respectively; simultaneous co-expression of BiP and PDI elevates the secretory expression level of recombinant human lysozyme by 50%.

BiP is the ER-resident homolog of cytoplasmic Hsp70. It stabilizes immature nascent proteins by binding to exposed hydrophobic peptide segments and maintains the structural stability of incompletely folded polypeptides.

In Pichia pastoris cell factories, engineered folding regulation via Hsp70 and Hsp40 chaperones can significantly enhance the biosynthesis efficiency of recombinant human interferon-γ. Single overexpression of Ydj1p (Hsp40), PDI and Ssa1p (Hsp70) achieves a nearly 4-fold increase in interferon-γ production; combined co-expression of Kar2p (BiP) and PDI produces a synergistic effect, boosting the target protein yield by approximately 6 times.

Molecular chaperones have achieved remarkable application achievements in large-scale recombinant protein production, while multiple challenges still remain to be addressed. Different types of proteins show distinct demand patterns for chaperone-assisted folding, hence it is essential to optimize the matching chaperone expression strategy for specific target proteins.

In addition, either excessive or insufficient expression of molecular chaperones will interfere with host cell growth metabolism and disrupt normal protein folding pathways, which requires precise quantitative regulation of intracellular chaperone expression levels. With continuous clarification of the molecular mechanism of chaperone-mediated protein folding, molecular chaperones will gain broader and more in-depth application prospects in the field of recombinant protein biomanufacturing.