Recombinant Protein Production: Comparing Systems at VectorBuilder and Overcoming Common Pitfalls
Scientist
on
August 19, 2024
This post was written by VectorBuilder, a global leader in gene delivery technologies. In this article, they compare different expression systems used for recombinant protein production, highlight common pitfalls and provide a preview of the next-generation strategies being employed at VectorBuilder. Their services are available on the Scientist.com marketplace.
Recombinant proteins are widely used as biological medicines and are utilized for a variety of research purposes. However, the production and purification of properly folded, high-yield recombinant proteins is usually not straightforward.
Options for Recombinant Protein Production
Bacterial expression: These are the most technically simple and cost-effective; in addition, they can be easily scaled up to achieve high yields. However, proteins produced in bacteria lack eukaryotic post-translational modifications that can be essential for biological activity, function and proper folding. Vectors must be designed carefully for bacterial expression as codon usage is different from eukaryotes, and therefore, codon optimization strategies can be used to improve expression.
Insect cell expression: Similar to bacterial expression systems, these cells are easy to scale up as they grow in high density suspension cultures and provide the added benefit of performing post-translational modifications and protein folding reflected in eukaryotic cells. Insect cells are also capable of producing secretory, intracellular and membrane-associated proteins and can also produce large protein complexes in tandem. However, this system is technically complex and time and labor-intensive.
Mammalian protein production: This system excels at generating mammalian proteins in their most natural state, complete with all necessary post-translational modifications and proper folding, making it ideal to produce a wide variety of proteins. However, long production times and complex, costly and difficult to scale up growth conditions can pose significant challenges to yields.
Cell-free protein production: Not relying on cell-specific production offers time-efficient synthesis, often completed within just three hours. This system is ideal for producing toxic, complex or unstable proteins. It is particularly suitable for high-throughput protein expression and screening due to its straightforward procedure, which is compatible with automated processes. Although optimizing conditions is relatively easy, this system is limited in the post-translational modifications it can provide.
Avoiding Common Pitfalls
Characterization of Recombinant Proteins
Proteins expressed in non-native environments often lack key post-translational modifications, like glycosylation, acetylation, phosphorylation and proper disulfide bond formation, which are critical for proper folding and biological activity. Without these modifications, proteins may misfold or fail to perform their desired biological effects. Thus, rigorous quality control is essential to characterize recombinant proteins, including biological activity testing, purity assessment via HPLC and even structural analysis through X-ray crystallography. VectorBuilder provides comprehensive protein characterization and functional validation to confirm the effectiveness of recombinant proteins in research applications.
Increasing Yields and Ensuring Proper Folding
Inclusion bodies are aggregates of misfolded or partially folded proteins that often form in bacteria when recombinant proteins are overexpressed, leading to lower yields. Extracting functional proteins from inclusion bodies involves complex purification processes including solubilization and refolding steps, which can be time-consuming and labor-intensive. Researchers can minimize the formation of inclusion bodies by optimizing expression conditions, such as lowering the temperature, reducing the expression rate with an inducible promoter or using a fusion tag to improve solubility. Additionally, choosing a more suitable host system can further reduce the risk of inclusion body formation.
Utilizing Next-Generation Strategies
Producing recombinant membrane-associated proteins is difficult due to their complex structure, folding pathways and requirements and hydrophobic regions that depend on membranes for proper functionality. When extracted from their native membrane environment, these proteins often lose their native conformation, making them difficult to purify in a functional state. Traditional purification methods can lead to misfolding or aggregation; therefore, they require specialized approaches such as using detergents, liposomes or nano-discs, to mimic the natural membrane environment during purification.
VectorBuilder has employed Virus-Like Particles (VLPs) to produce recombinant proteins and membrane-associated proteins in their native conformation. VLPs are non-infectious particles composed of a viral shell but lacking a viral genetic material. Membrane Protein VLPs (MP-VLPs) are a subtype of VLPs produced by co-expressing a retroviral structural core protein (gag) with the target membrane protein in mammalian cells. This method ensures that membrane-associated proteins are incorporated in their native conformation, making MP-VLPs a valuable tool for researchers needing properly folded and functional membrane proteins.
Conclusion
Choosing the right system and strategy for recombinant protein production can be daunting, given the numerous considerations and options. Each system has unique strengths and challenges, with the best choice depending on factors such as post-translational modifications, proper folding, yields, scalability and the protein’s intended use. VectorBuilder provides comprehensive recombinant protein production services, helping researchers navigate and overcome these challenges effectively. To get started visit their Recombinant Protein webpage.