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Recombinant Proteins: How Natural Proteins Are Engineered for Research & Therapy
Biointron2025-02-28Read time: 10 mins
Recombinant proteins: useful reagents for many different applications. DOI: 10.1186/s12934-022-01778-5
Recombinant proteins have applications in biotechnology from basic research to diagnostics and therapeutics. With genetic engineering, recombinant protein technology allows control over protein expression, structure, and function. This approach facilitates large-scale production, protein modifications, and the development of proteins with enhanced or novel properties.
What Are Recombinant Proteins?
Proteins are linear chains of amino acids that fold into three-dimensional structures to perform various cellular functions, including enzymatic activity, structural support, and immune defense. The sequence of a protein is dictated by the genetic code, which is transcribed from DNA into messenger RNA (mRNA) and subsequently translated into protein by ribosomes.
Recombinant proteins are distinct from natural proteins in that they are produced using genetically engineered DNA in a controlled expression system. This allows researchers to:
Express proteins in host cells that do not naturally produce them.
Modify protein sequences to improve stability, solubility, or functionality.
Enable large-scale production for industrial and clinical applications.
The production of recombinant proteins begins with the isolation of the gene encoding the protein of interest. The gene may be modified to optimize expression, and it is inserted into an expression vector. This vector is introduced into a host cell, which serves as a factory for protein production. After expression, the recombinant protein is purified and characterized for downstream applications.
General considerations for recombinant proteins production. DOI: 10.53560/PPASB(60-1)731
Key Components of Recombinant Protein Production
The production of recombinant proteins requires several essential elements:
1. Genetic Material
The gene encoding the target protein is obtained either from a natural source, such as a cell or organism, or synthesized in vitro. Synthetic gene synthesis allows for codon optimization, which can enhance protein expression in a specific host.
2. Expression Vectors
Expression vectors are DNA constructs that carry the gene of interest along with regulatory elements that control protein expression. These include:
Promoters: Sequences that initiate transcription. Strong promoters enhance protein production.
Selection Markers: Genes that confer antibiotic resistance or other selectable traits, allowing for the identification of successfully transformed/transfected cells.
Fusion Tags: Additional sequences that facilitate protein purification, such as His-tag, GST-tag, or FLAG-tag.
3. Host Cells
The choice of host cell system significantly affects protein yield, post-translational modifications, and production scalability. Common host systems include:
Bacterial Cells (e.g., Escherichia coli): High yield, cost-effective, and fast-growing. Lacks post-translational modifications, making it unsuitable for complex eukaryotic proteins. Risk of protein misfolding and aggregation into inclusion bodies.
Mammalian Cells (e.g., CHO, HEK293): Capable of producing proteins with correct post-translational modifications. Essential for therapeutic proteins such as monoclonal antibodies. Expensive and slower-growing compared to bacteria.
Yeast and Insect Cells (e.g., Pichia pastoris, Baculovirus-infected insect cells): Provide post-translational modifications similar to mammalian systems. Higher yields compared to mammalian expression but may exhibit different glycosylation patterns.
4. Growth Media
Host cells require a nutrient-rich environment for optimal growth and protein production. Growth media contain carbon sources, nitrogen, salts, and trace elements necessary for cellular metabolism. Mammalian cell cultures often require specialized media supplemented with growth factors.
5. Protein Purification Systems
Once expressed, recombinant proteins must be isolated from host cells and purified for downstream applications. Common purification techniques include affinity chromatography, ion-exchange chromatography, and size-exclusion chromatography.
Steps in Recombinant Protein Production
General methodology for recombinant protein production in expression system and afterward purification. DOI: 10.53560/PPASB(60-1)731
1. Construction of Recombinant DNA
The first step in recombinant protein production involves engineering recombinant DNA that encodes the target protein. This can be achieved through:
Gene Cloning: Extracting the gene from genomic or complementary DNA (cDNA) sources.
Gene Synthesis: Artificially synthesizing the gene sequence with optimized codons for improved expression in a specific host.
Vector Construction: Inserting the gene into a suitable expression vector containing regulatory elements.
Once the recombinant DNA construct is ready, it is introduced into host cells through:
Transformation (Bacteria & Yeast): Uptake of foreign DNA by cells, often using heat shock or electroporation.
Transfection (Mammalian Cells): Introduction of recombinant DNA using lipid-based reagents, electroporation, or viral vectors.
2. Protein Expression and Post-Translational Modifications
Following successful gene integration, the host cells translate the mRNA into protein. Several factors influence protein expression:
Codon Optimization: Enhances translation efficiency in the chosen host system.
Protein Folding and Solubility: Chaperones and folding catalysts help prevent misfolding and aggregation.
Post-Translational Modifications: Essential for the function of many therapeutic proteins, including glycosylation, phosphorylation, and disulfide bond formation.
Mammalian cells provide the most accurate modifications, making them the preferred choice for therapeutic proteins such as monoclonal antibodies and cytokines.
3. Downstream Processing: Purification and Quality Control
Following expression, recombinant proteins must be extracted and purified from the host cells. This process involves:
A. Cell Lysis
Depending on the host cell type, different lysis methods are used to release the recombinant protein:
Applications of Recombinant Proteins in Biotechnology
Recombinant protein technology has broad applications in various fields:
Therapeutic Proteins: Monoclonal antibodies, insulin, growth factors, and cytokines are produced recombinantly for medical use.
Vaccine Production: Recombinant proteins serve as vaccine antigens (e.g., hepatitis B surface antigen, SARS-CoV-2 spike protein).
Enzyme Replacement Therapy: Treats metabolic disorders like Gaucher’s disease and Pompe disease.
Industrial Enzymes: Used in biofuel production, detergent formulations, and food processing.
Diagnostic Tools: Recombinant proteins serve as antigens in serological tests and immunoassays.
Recombinant protein technology continues to advance, with innovations in gene editing, protein engineering, and expression systems improving yield, functionality, and therapeutic potential. As the demand for high-quality recombinant proteins increases, optimization of production and purification strategies remains a critical focus in biotechnology and pharmaceutical industries.
Advances in Bioprocessing for Large-Scale Recombinant Protein Production
The large-scale production of recombinant proteins is critical for meeting the global demand for biopharmaceuticals, vaccines, and diagnostic reagents. With infectious diseases and cancers being major causes of mortality worldwide, the ability to efficiently produce therapeutic proteins at an industrial scale has become a priority. Advances in recombinant DNA technology and bioprocessing strategies have significantly improved the yield, cost-efficiency, and scalability of recombinant protein production. Recent developments include high-throughput process development, single-use systems, continuous bioprocessing, and regulatory-driven Quality by Design (QbD) approaches, all of which enhance biopharmaceutical manufacturing.
High-Throughput Process Development (HTPD) in Bioprocess Optimization
Optimization of recombinant protein production requires evaluation of various factors, including media composition, pH, aeration, temperature, and induction parameters. Traditional optimization methods are time-consuming and resource-intensive. High-throughput process development (HTPD) has emerged as a powerful approach to accelerate optimization using automated systems and miniaturized bioreactors.
HTPD enables:
Parallel screening of multiple conditions to rapidly determine the optimal expression parameters.
Data-driven decision-making through integration with bioinformatics and machine learning models.
Cost-effective process development by reducing material and time requirements compared to traditional screening methods.
HTPD has been particularly beneficial in optimizing mammalian cell cultures used for monoclonal antibody (mAb) production, where small variations in process conditions can significantly affect protein quality and yield.
Single-use bioreactors and disposable components have revolutionized bioprocessing by reducing operational costs, minimizing contamination risks, and increasing flexibility. These systems replace traditional stainless-steel bioreactors and chromatography columns with pre-sterilized, disposable components, eliminating the need for extensive cleaning and validation between production batches.
Traditional batch and fed-batch cultures have been the mainstay of recombinant protein production; however, continuous bioprocessing is gaining traction as a method to improve productivity and efficiency. Continuous bioprocessing integrates upstream and downstream processing into a seamless, uninterrupted workflow.
Application of high throughput process development in bioprocessing. DOI: 10.1016/j.coche.2014.08.004
Advancements in Continuous Upstream Processing
Perfusion bioreactors are a key component of continuous upstream processing. Unlike fed-batch systems, which involve periodic nutrient additions, perfusion systems continuously supply fresh media while removing waste products. This ensures a stable environment for recombinant protein production. Perfusion culture has been particularly effective for producing monoclonal antibodies, as it enhances cell viability and prolongs protein expression.
Innovations in Continuous Downstream Processing
Continuous chromatography and viral inactivation methods are transforming downstream processing, enabling real-time purification and quality control. Innovations include:
Multicolumn continuous chromatography, which improves purification efficiency and reduces buffer consumption.
Automated viral clearance steps, ensuring regulatory compliance while maintaining process continuity.
Inline process analytical technology (PAT) for real-time monitoring of product purity and yield.
The integration of continuous upstream and downstream processing remains a challenge, but advances in process modeling and automation are driving progress toward fully continuous biomanufacturing.
Future Directions in Biopharmaceutical Manufacturing
As the demand for recombinant proteins continues to grow, the focus is shifting toward:
Further integration of artificial intelligence and machine learning for predictive process control.
Expansion of glycoengineering techniques to enhance the biological activity of therapeutic proteins.
Development of fully automated and self-regulating bioprocessing platforms to minimize human intervention.
The transition toward fully continuous, integrated bioprocessing is expected to redefine large-scale production, offering cost savings and increased efficiency in the manufacture of recombinant protein-based biopharmaceuticals.
References:
Tripathi, N. K., & Shrivastava, A. (2019). Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development. Frontiers in Bioengineering and Biotechnology, 7, 420. https://doi.org/10.3389/fbioe.2019.00420
