Comparison of Protein Expression Systems

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Protein expression systems are central to biotechnology, enabling the production of recombinant proteins for a wide range of applications, from industrial enzymes and diagnostics to pharmaceuticals and academic research. At Creative Biostructure we offer protein expression services using various expression systems. Here we provide a detailed comparison of different protein expression systems, examining recombinant protein expression methods, factors influencing host system selection, and the characteristics of cell-free, bacterial, yeast, mammalian, and insect expression systems. Understanding the unique advantages and limitations of each will help you choose the optimal solution for your project.

Recombinant Protein Expression Methods

Recombinant protein expression involves the introduction of a gene of interest into a host system capable of transcribing and translating it into the desired protein. This process typically involves gene cloning, transformation/transfection, expression induction, protein folding and modification, and purification. For more information, please visit the basics of protein expression page.

Key steps in recombinant protein expression: gene cloning, transformation/transfection, expression induction, protein folding and modification, and purification. Figure. 1: Process of recombinant protein expression. (Created with BioRender.com)

Critical Parameters to Select a Host System

Selecting a host system for protein expression is a multifaceted decision influenced by several critical parameters:

Protein Complexity: The structural and functional complexity of the target protein dictates the need for advanced PTMs, proper folding, and multi-subunit assembly. For simple proteins, bacterial systems may suffice, while complex proteins require eukaryotic hosts.
Post-Translational Modifications: PTMs such as glycosylation, phosphorylation, acetylation, and disulfide bond formation are critical for many eukaryotic proteins. Bacterial systems lack these capabilities, whereas mammalian systems provide the most authentic PTMs.
Yield Requirements: High yield is crucial for industrial applications. Bacterial and yeast systems excel at producing large quantities of protein, often at the expense of PTM fidelity.
Protein Stability and Activity: Host systems differ in their ability to produce stable, bioactive proteins. Authentic folding and modifications are essential for therapeutic and diagnostic proteins.
Production Speed: Time constraints favor fast-growing systems such as bacteria and yeast, while mammalian and insect systems typically require longer development cycles.
Scalability: Industrial-scale protein production requires host systems that are easily scalable. Bacteria and yeast systems offer easy scalability.
Cost Constraints: Budget limitations often dictate the choice of a cost-effective host system, with bacterial systems being the least expensive and mammalian systems being the most resource intensive.
Protein Toxicity: Proteins that are toxic to the host cell may require the use of specialized systems, such as cell-free expression or tightly controlled inducible systems.

Comparison of Protein Expression Systems

Recombinant protein expression technology enables a variety of downstream applications, including the analysis of gene regulation, protein structure and function, protein-protein interactions, and drug discovery and development. Using the right protein expression system for your specific application can be critical to your success. The following table summarizes the benefits and challenges of each protein expression system:

Host System	Advantages	Challenges
Bacterial	High Efficiency: Bacteria grow rapidly and can reach high densities, leading to quick protein production. Cost-Effectiveness: Minimal culture requirements and inexpensive media reduce production costs. Easy Manipulation: Genetic engineering in bacteria is straightforward and well-established.	Lack of PTMs: Bacteria cannot perform eukaryotic PTMs, limiting their ability to produce functional eukaryotic proteins. Inclusion Bodies: Proteins expressed in bacteria may aggregate into insoluble inclusion bodies, necessitating complex refolding protocols. Limited Protein Secretion: Secretion pathways are inefficient in bacteria, requiring proteins to be purified from intracellular extracts.
Mammalian	Human-Like PTMs: Mammalian cells perform authentic PTMs, including complex glycosylation and phosphorylation. High Bioactivity: Proteins expressed in mammalian systems exhibit native folding, stability, and functionality. Versatility: Capable of producing antibodies, cytokines, and complex membrane proteins.	Cost: High media and infrastructure costs make mammalian systems expensive. Long Production Time: Slow growth rates increase production timelines. Complex Scalability: Large-scale manufacturing requires advanced bioreactor technology and expertise.
Yeast	Eukaryotic Machinery: Yeast can perform PTMs such as glycosylation and disulfide bond formation. High Yield: Yeast can grow to high cell densities, making it suitable for large-scale production. Cost-Effectiveness: Cultivation is relatively inexpensive compared to mammalian systems. Secretion Capability: Many yeast systems are optimized for efficient protein secretion, simplifying purification.	Eukaryotic Machinery: Yeast can perform PTMs such as glycosylation and disulfide bond formation. High Yield: Yeast can grow to high cell densities, making it suitable for large-scale production. Cost-Effectiveness: Cultivation is relatively inexpensive compared to mammalian systems. Secretion Capability: Many yeast systems are optimized for efficient protein secretion, simplifying purification.
Insect	Eukaryotic PTMs: Insect cells can perform many PTMs, including disulfide bond formation and glycosylation. High Yield: Baculovirus infection leads to robust protein production. Faster Development: Production timelines are shorter than mammalian systems.	Non-Human Glycosylation: Glycosylation patterns differ from those in humans, which can affect functionality in therapeutic contexts. Viral Handling: Requires careful management of baculovirus stocks and infection cycles.
Cell-Free	Non-Human Glycosylation: Glycosylation patterns differ from those in humans, which can affect functionality in therapeutic contexts. Viral Handling: Requires careful management of baculovirus stocks and infection cycles.	High Cost: Cell-free systems are costly due to the preparation of cell lysates and the need for expensive reagents. Low Yield: While scalable, yields are generally lower compared to living cell systems. Limited PTMs: The ability to perform complex PTMs depends on the origin of the cell extract and is often less efficient than in living systems.

In a review of guidelines for selecting appropriate gene expression systems for recombinant protein production, the most commonly used and accessible systems were visually scored according to 7 important parameters.

Figure. 2: Comparative overview of the characteristics associated with the major gene expression systems. Scoring: Ease of use: (1) Possible with SOP + user training + 1 year hands-on experience; (2) possible with SOP + user training + short-term experience; (3) possible with SOP + user training; (4) possible with SOP+ short-term experience; (5) possible with SOP only. Speed: (1) >8 weeks; (2) 4-8 weeks; (3) 1-4 weeks; (4) 3-7 days; (5) 1-3 days. Protein production capacity: (1) <1 mg/L; (2) 1-5 mg; (3) 5-20 mg/L; (4) 20-100 mg/L; (5) >100 mg/L. Protein secretion: (1) <1 mg/L; (2) 1-5 mg/L; (3) 5-20 mg/L; (4) 20-100 mg/L; (5) >100 mg/L. Protein folding and assembly-size: (1) <50 kDa; (2) 50-100 kDa; (3) 100-250 kDa; (4) 250-500kDa; (5) >500 kDa. Protein folding and assembly—SS bonds: (1) 1 SS bond; (2) 2 SS bonds; (3) 3-4 SS bonds; (4) 5-10 SS bonds; (5) >10 SS bonds. Cost efficiency: (5) <50 €/L; (4) 50-100 €/L; (3) 100-500 €L; (2) 500-1,000 €ЛL; (1) >1,000 €/L. (Schütz A et al., 2023)

Protein expression systems are essential tools tailored to meet the diverse needs of research and industry. Creative Biostructure offers customized solutions for all major expression systems—bacterial, yeast, mammalian, insect and cell-free—to ensure that your research or industrial goals are achieved with precision and efficiency. Contact us today and improve your protein expression capabilities!

Reference

Schütz A, Bernhard F, Berrow N, et al. A concise guide to choosing suitable gene expression systems for recombinant protein production. STAR Protocols. 2023;4(4):102572.