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Co-expression of Protein Complexes: Strategies, Challenges, and Applications

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Protein complexes play essential roles in virtually all cellular processes, from signal transduction to structural integrity. Understanding their assembly and function is critical for fields such as molecular biology, drug discovery, and structural biology. Co-expression, a technique that allows the simultaneous production of multiple proteins in a single host system, has become a powerful tool for studying protein complexes.

What is Co-expression?

Co-expression is the process of simultaneously producing two or more proteins in a single host organism. This technique is an important tool in molecular biology and biotechnology, allowing researchers to more accurately model and study complex biological systems. The ability to express multiple proteins in parallel enables the study of their interactions and functions in a controlled environment, facilitating a deeper understanding of cellular processes, protein function, and the formation of protein complexes.

By using co-expression, researchers can simulate biological conditions in which proteins naturally work together, as opposed to isolating individual proteins. This ability to replicate complex biological interactions is critical to advancing research in a variety of fields, including drug discovery, structural biology, and synthetic biology.

Why is Co-expression Necessary?

Co-expression is particularly important because many proteins only function when they are part of a larger, multi-protein complex. Isolating individual proteins can lead to misfolding, degradation, and loss of function. When proteins are expressed individually, they can face a number of challenges that interfere with their functionality. These challenges include

  • Misfolding: When proteins are expressed alone, they may not fold into their correct 3D structures. Many proteins require specific interactions with other proteins or cofactors for proper folding. Without these interactions, they may remain in an unfolded or improperly folded state, rendering them non-functional.
  • Degradation: Isolated proteins that are misfolded or incomplete are often recognized and degraded by the cell's quality control mechanisms. This reduces the overall yield of the protein and complicates subsequent analyses.
  • Loss of Function: Proteins that need to interact with other molecules for their activity can lose their function when expressed in isolation. Co-expression provides the necessary interaction partners to ensure that proteins retain their biological activity.

Co-expression addresses these issues by enabling:

  • Proper Folding and Assembly: In co-expression systems, proteins are expressed together, allowing them to interact and fold correctly as they would in a cellular environment. This improves the efficiency and quality of protein production.
  • Enhanced Solubility: Co-expression often leads to increased solubility of proteins because the interaction between protein subunits can stabilize their structure. This reduces the risk of aggregation, a common problem when proteins are expressed individually.
  • Simultaneous Post-Translational Modifications: Many proteins require post-translational modifications (PTMs) such as phosphorylation, glycosylation, or acetylation for their functionality. Co-expression allows these modifications to occur simultaneously, ensuring that the proteins are in their native, functional state.

Co-expression Strategies

Choice of Expression System

Selecting the right expression system is crucial for successful co-expression, as the system should support the desired protein interactions and modifications. Different expression systems have their own advantages and limitations:

  • Bacterial Systems: Bacteria, such as Escherichia coli, are commonly used for co-expression because they are cost-effective and easy to manipulate. They grow rapidly, which is ideal for expressing simple protein complexes. However, bacteria lack the advanced eukaryotic post-translational modification machinery, so they may not be suitable for expressing more complex proteins that require specific modifications.
  • Yeast Systems: Yeast offers a middle ground by providing some eukaryotic post-translational modification capabilities, making it useful for moderately complex proteins. Yeast systems are often used for expressing membrane proteins and some enzyme complexes.
  • Insect Cells: The baculovirus expression system used in insect cells is highly efficient for the production of eukaryotic proteins. This system supports proper protein folding and modification, including glycosylation, and can be scaled for high yields. Insect cells are particularly useful for expressing larger or more complex protein assemblies.
  • Mammalian Systems: Mammalian cells are ideal for producing complex proteins that require specific eukaryotic modifications, such as complex glycosylation patterns or disulfide bonds. While they provide the most accurate and biologically relevant expression system, mammalian cells are slower and more expensive to culture than bacterial or yeast systems.

Vector Design

The design of the expression vector plays a crucial role in ensuring the successful co-expression of proteins. There are two primary strategies for vector design:

  • Single-Vector Systems: In a single vector system, all the genes for the proteins to be co-expressed are cloned into a single plasmid. This method simplifies the transformation process because only one plasmid needs to be introduced into the host. However, the challenge is to select appropriate promoters and control elements to ensure that the proteins are expressed in the correct proportions.
  • Multi-Vector Systems: In a multi-vector system, each protein is carried on a separate plasmid. This allows for more flexibility, as researchers can independently control the expression levels of each protein. However, this system requires co-transformation of multiple plasmids, which can be more complex.

Diagram of expression vectors and co-expression strategies in E. coli, illustrating multiple and single vector approaches.Figure 1. Definition of expression vectors and co-expression strategies. The diagram depicts expression vectors and strategies for co-expression in E. coli. Ovals represent E. coli cells, and rectangles represent expression vectors. Bent arrows represent individual expression cassettes, and shaded rectangles are for individual ORFs. The rounddots represent either ribosome binding sites or linker sequences, as indicated. Each vector can be used alone or in combination with others. SD, Shine Dalgarno sequence; IRES, internal ribosome entry site; ORF, open reading frame. (Kerrigan et al., 2011)

Full Length vs. Truncations/Deletions/Mutations

Full-length proteins are often preferred to preserve native folding, post-translational modifications (PTMs), and interaction domains. For example, co-expression of full-length receptors with their ligands ensures biologically relevant binding kinetics. However, challenges such as insolubility or misfolding in heterologous systems may require truncation. Removal of non-essential domains (e.g. transmembrane regions or intrinsically disordered segments) can improve solubility and facilitate purification. For example, truncation of the cytoplasmic domain of a membrane protein allows its soluble expression in E. coli.

Deletions and mutations are used to dissect functional domains or mimic pathological variants. Co-expression of wild-type and mutant proteins (e.g. oncogenic Ras mutants with downstream effectors) can reveal dominant-negative effects or compensatory interactions. However, truncations risk disrupting quaternary structures, as seen in misfolded antibody fragments lacking constant regions.

Tagging Options

Tags are critical for detection, purification, and functional studies. Common choices include:

  • Affinity tags (His, GST, MBP): Facilitate purification via immobilized metal or glutathione resins.
  • Epitope tags (FLAG, HA, Myc): Enable immunodetection in Western blots or pull-down assays.
  • Fluorescent tags (GFP, mCherry): Allow real-time tracking of co-localization or interaction via Förster resonance energy transfer (FRET).
  • Self-cleaving tags (TEV protease site): Permit tag removal post-purification to avoid steric hindrance.

Tag placement (N- vs. C-terminal) must be carefully optimized to avoid interfering with functional domains, such as ligand binding sites or membrane anchoring regions. For example, N-terminal His tags on GPCRs may interfere with ligand binding, while C-terminal fusions may interfere with membrane anchoring. Multi-tag systems (e.g. His-Strep II dual tags) allow sequential purification of co-expressed partners.

Stable vs. Transient Expression

Stable expression involves the integration of transgenes into the host genome, ensuring long-term protein production. This is ideal for large-scale applications (e.g., industrial enzyme production) or for studying chronic interactions (e.g., viral latency factors in host cells). However, the generation of stable lines (e.g. HEK293 or CHO cells) is time-consuming and requires antibiotic selection or fluorescence-activated cell sorting (FACS).

Transient expression uses episomal vectors (e.g., plasmid DNA or viral vectors) for rapid, high-yield protein synthesis within 24–72 hours. It is advantageous for screening multiple constructs (e.g., co-expression of kinase mutants with substrates) or toxic proteins. However, transient systems suffer from batch-to-batch variability and short-term viability, limiting their use in longer-term studies.

Homologous Expression vs. Heterologous Expression

Homologous expression refers to the production of proteins in their native host. This preserves PTMs (e.g. glycosylation in mammalian proteins) and avoids codon bias problems. For example, co-expression of human chaperones with their client proteins in HEK293 cells ensures proper folding. However, homologous systems may lack scalability or genetic tractability.

Heterologous expression uses non-native hosts (e.g., E. coli, yeast, or insect cells) for low-cost, high-yield production. E. coli is widely used for the co-expression of prokaryotic proteins (e.g., bacterial toxin-antibody systems), but often fails to process eukaryotic PTMs. Baculovirus/insect cell systems bridge this gap by allowing glycosylation and disulfide bond formation, making them ideal for antibody-antigen co-expression.

Challenges in heterologous systems include codon bias (mitigated by codon optimized genes), improper protein folding (addressed by chaperone co-expression), and incompatible PTMs. For example, co-expression of human kinases in yeast may require exogenous phosphorylation machinery.

Co-transformation vs. Co-infection

Co-expression can be achieved through different methods depending on the expression system being used:

  • Co-transformation: This method involves the simultaneous introduction of multiple plasmids into a host organism and is commonly used in bacterial and yeast systems. Co-transformation allows the expression of multiple proteins in the same host, although careful optimization is required to ensure that each protein is produced at the desired level.
  • Co-infection: In insect cell systems using the baculovirus expression system, separate viruses are used to deliver each gene into the host cells. This allows for the expression of multiple proteins in the same cell without the need to combine plasmids. Co-infection is particularly advantageous for complex protein complexes that require the coordinated expression of multiple genes.

Flowchart of key factors in designing a co-expression strategy, covering purpose, protein complex, partners, modifications, co-expression and purification.Figure 2. Flowchart of critical considerations in designing a co-expression strategy. A purpose driven flowchart is shown with critical steps involved in designing a co- expression strategy outlined. The decision of choosing a specific partner(s) for co-expression is a result of database searches and literature study of the target and its biology, together with a judgment on using a relevant splice variant (if any). The expression vectors could be any one or more of the vectors with a single or multiple expression cassettes, polycistronic, and/or single chain fusions. The host could be cells of mammalian origin, or derived from insect, yeast, or bacteria. (Adapted from Kerrigan et al., 2011)

Challenges in Co-expression

  • Stoichiometry Issues: Maintaining natural protein ratios is critical. Over- or underexpression of a subunit can lead to misassembly, aggregation, or non-functional complexes.
  • Post-translational Modifications: Many eukaryotic proteins require modifications such as glycosylation or phosphorylation, which are difficult to achieve in bacterial systems. This limitation necessitates the use of yeast, insect, or mammalian cells.
  • Toxicity: Some proteins or their intermediates can be toxic to the host, requiring tightly controlled expression systems to minimize host cell stress or death.
  • Solubility: Misfolded or overexpressed proteins often aggregate, making it essential to optimize conditions for proper folding and solubility.

Applications of Protein Complex Co-expression

Structural Biology

Stable and functional protein complexes are essential for structural studies because they provide insight into molecular interactions and biological mechanisms. Techniques such as cryo-electron microscopy (cryo-EM), X-ray crystallography, and nuclear magnetic resonance (NMR) spectroscopy require well-formed protein assemblies to determine high-resolution structures. Co-expression allows the simultaneous production of interacting proteins, facilitating the assembly of complete complexes required for structural studies. This approach helps scientists understand protein folding, binding sites, and conformational changes critical for cellular function.

Drug Discovery

Protein co-expression is a powerful tool in pharmaceutical research, particularly for target validation and inhibitor screening. Many therapeutic targets, including receptors, enzymes, and signaling proteins, function as complexes. By co-expressing these proteins, researchers can recapitulate their natural interactions in vitro, enabling precise drug binding studies. This approach improves the screening of small molecules, peptides, and antibodies for drug development. In addition, co-expression aids in the structural analysis of protein-drug complexes, guiding rational drug design and optimization.

Synthetic Biology

Synthetic biology relies heavily on co-expression to construct artificial biological pathways for industrial and pharmaceutical applications. By engineering multiple genes within a host organism, researchers can create biosynthetic pathways that produce valuable compounds such as antibiotics, vitamins, and biofuels. For example, co-expression of multiple enzymes in a pathway enables the efficient conversion of precursor molecules into target products, increasing yield and process efficiency. This strategy is critical to the development of novel biomanufacturing techniques that replace traditional chemical synthesis with sustainable, bio-based alternatives.

Industrial Biotechnology

In industrial biotechnology, functional protein complexes are used in large-scale biocatalysis for manufacturing, waste treatment and sustainable production processes. Multi-enzyme systems produced by co-expression enable efficient and selective biochemical reactions in industries such as food processing, bioenergy and pharmaceuticals. For example, enzyme cascades co-expressed in microbial hosts can break down lignocellulosic biomass into fermentable sugars for biofuel production. Similarly, co-expressed enzyme systems increase the efficiency of bioremediation processes by breaking down contaminants into harmless compounds. By leveraging co-expression, industries can improve process efficiency, reduce energy consumption and minimize environmental impact.

Case Studies

Case Study 1: Protein Co-Expression Analysis as a Strategy to Complement a Standard Quantitative Proteomics Approach: Case of a Glioblastoma Multiforme Study

Although underutilized in proteomics, protein co-expression analysis provides unique insights beyond conventional methods. This study applies weighted gene co-expression network analysis to a glioblastoma multiforme proteomic dataset and identifies three major modules associated with membrane-associated groups: mitochondrial, endoplasmic reticulum, and vesicle fractions. These networks were validated against the STRING database and showed significant overlap.

Further analysis de-clustered the modules into smaller networks based on connectivity, hierarchical clustering, and Gene Ontology enrichment. The endoplasmic reticulum module highlighted redox activity, unfolded protein response elements, and oxidative stress pathways. In the mitochondrial module, proteins of complex I of the electron transfer chain were downregulated more than those of complex III. In addition, the two pyruvate kinase isoforms exhibited distinct co-expression patterns, suggesting functional roles in separate cellular locations. This approach reveals complex protein interactions and regulatory mechanisms in glioblastoma.

Protein co-expression analysis as a complementary strategy in quantitative proteomics: A case study of glioblastoma multiforme.Figure 3. On the left, the step-wise protein list fragmentation is illustrated. On the right, the different bioinformatics tools used are described. The number of proteins per group are presented and colored using the WGCNA color coding. The names associated to the sub-clusters are illustrated on the right side of each sub-Clusters. (Kanonidis et al., 2016)

Case Study 2: Expanding the landscape of recombinant protein production in Escherichia coli

Advances in vectors and host strains have improved the overexpression of recombinant proteins in Escherichia coli. Recently, co-expression strategies using single vectors or multiple compatible plasmids have gained attention. Initially developed for the production of protein complexes in vivo, co-expression has expanded the utility of E. coli as a platform for heterologous protein production. It facilitates the synthesis of protein complexes, proteins with post-translational modifications, and unnatural amino acids. Co-expression also supports efficient recombinant protein secretion, including overproduction of membrane proteins. For example, co-expression of HlyB and HlyD secretion factors allows extracellular overexpression of metalloprotease.

Diagram of recombinant protein co-expression and secretion in E. coli using type I and II secretion systems for metalloprotease and chitinase production via compatible plasmids and transporters.Figure 4. Co-expression of genes and secretion of recombinant proteins in E. coli. The type I secretion system was used in E. coli to produce a metalloprotease from S. marcescens. This protease was overexpressed from of a ColE1-type vector, and the HlyB and HlyB hemolysin transporters were co-expressed by means of a compatible p15A-type plasmid. The endogenous TolC transporter afforded secretion of the metalloprotease into the extracellular medium. The secretion of chitinase, overexpressed in E. coli from a pUC derivative, was obtained co- expressing (using a compatible p15A-type vector) the Gsp locus, coding for a type II secretion system. The overexpressed chitinase, bearing a signal sequence (blue sphere) is transported across the inner membrane via the Sec translocon. Subsequently, the signal peptide is cleaved and the chitinase secreted through the GspCD transporter. (Hochkoeppler, 2013)

Future Perspectives

  • Advanced Expression Systems: Emerging technologies, such as cell free expression systems, promise higher yields and faster turnover for co-expression studies.
  • Artificial Intelligence: Machine learning models are being developed to predict optimal co-expression conditions, including stoichiometry and host selection.
  • Expanded Applications: As co-expression methodologies improve, their applications will likely extend further into personalized medicine and regenerative therapies.

The co-expression of protein complexes is an indispensable tool in modern biology and biotechnology. By understanding the strategies, overcoming the challenges, and learning from case studies, researchers can unlock new insights into the functional mechanisms of protein assemblies. As the field advances, the potential applications of co-expression will continue to grow, driving innovation across scientific disciplines.

These protein co-expression case studies showcase successful applications and strategies tailored to different research needs. At Creative Biostructure, we offer a comprehensive suite of protein expression services designed to meet the unique needs of our customers, from bacterial expression to cell free expression. Our expert team is dedicated to delivering customized solutions with precision and efficiency. For more information or to discuss your specific needs, please contact us.

References

  1. Kanonidis EI, Roy MM, Deighton RF, Le Bihan T. Protein co-expression analysis as a strategy to complement a standard quantitative proteomics approach: case of a glioblastoma multiforme study. Rabilloud T, ed. PLoS ONE. 2016;11(8):e0161828.
  2. Kerrigan JJ, Xie Q, Ames RS, Lu Q. Production of protein complexes via co-expression. Protein Expression and Purification. 2011;75(1):1-14.
  3. Hochkoeppler A. Expanding the landscape of recombinant protein production in Escherichia coli. Biotechnol Lett. 2013;35(12):1971-1981.
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