6.2 Protein expression systems
3 min read•august 7, 2024
Protein expression systems are crucial for producing recombinant proteins in various organisms. From simple bacteria to complex mammalian cells, each system offers unique advantages for different protein types and applications. Understanding these systems is key to successful protein production and engineering.
Choosing the right expression system depends on the protein's complexity and intended use. Prokaryotic systems like E. coli are great for simple proteins, while eukaryotic systems like yeast and mammalian cells handle complex proteins with post-translational modifications. Cell-free systems offer rapid production for special cases.
Prokaryotic and Cell-free Expression Systems
Prokaryotic Expression Systems and Escherichia coli
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- commonly used for protein production due to their simplicity, rapid growth, and high yields
- Escherichia coli (E. coli) most widely used prokaryotic host for recombinant protein expression
- Well-characterized genetics and physiology
- Easily manipulated and cultured in large quantities
- Capable of producing high levels of recombinant proteins
- Limitations of E. coli expression systems include inability to perform post-translational modifications and potential formation of inclusion bodies
Cell-free Systems and Codon Optimization
- Cell-free systems allow protein synthesis without the use of living cells by utilizing cell extracts containing necessary components for transcription and translation
- Advantages include rapid protein production, easy manipulation of reaction conditions, and ability to express toxic or membrane proteins
- Cell-free systems derived from various sources (E. coli, wheat germ, rabbit reticulocytes)
- Codon optimization involves altering the codons in a gene sequence to match the codon usage preference of the host organism
- Improves protein expression levels by increasing translation efficiency
- Achieved through site-directed mutagenesis or gene synthesis
Inducible Promoters in Prokaryotic Expression
- Inducible promoters allow controlled expression of recombinant proteins in response to specific stimuli or inducing agents
- Commonly used inducible promoters in E. coli include:
- lac promoter induced by lactose or its analog IPTG (isopropyl β-D-1-thiogalactopyranoside)
- T7 promoter recognized by T7 RNA polymerase and induced by IPTG
- ara promoter induced by arabinose
- Inducible promoters enable tight regulation of protein expression, minimizing potential toxic effects on host cells and allowing optimization of expression conditions
Eukaryotic Expression Systems
Yeast Expression Systems
- Yeast expression systems combine the advantages of prokaryotic and eukaryotic systems
- Saccharomyces cerevisiae (baker's yeast) and Pichia pastoris commonly used yeast hosts
- Capable of performing post-translational modifications similar to higher eukaryotes
- Easily cultured and manipulated, with rapid growth rates
- Suitable for large-scale fermentation and high-density cell culture
- Yeast expression systems particularly useful for producing secreted proteins and glycoproteins
Mammalian Cell Expression Systems
- Mammalian cell expression systems provide the most authentic environment for producing complex eukaryotic proteins
- Common mammalian cell lines used include CHO (Chinese Hamster Ovary), HEK293 (Human Embryonic Kidney), and NS0 (mouse myeloma) cells
- Capable of performing human-like post-translational modifications, including glycosylation and proper protein folding
- Suitable for producing and monoclonal antibodies
- Mammalian cell culture requires more complex media and growth conditions compared to prokaryotic and yeast systems
- Transient or stable transfection methods used to introduce recombinant DNA into mammalian cells
Baculovirus Expression System
- Baculovirus expression system utilizes insect cells infected with recombinant baculovirus for protein production
- Autographa californica multiple nucleopolyhedrovirus (AcMNPV) most commonly used baculovirus vector
- Large cloning capacity allows insertion of large genes or multiple genes
- Strong polyhedrin promoter drives high-level expression of recombinant proteins
- Insect cells (Sf9 or High Five) used as hosts for baculovirus infection and protein expression
- Capable of performing post-translational modifications similar to mammalian cells
- Suitable for producing complex proteins, including membrane proteins and virus-like particles (VLPs)
Key Terms to Review (18)
Affinity Chromatography: Affinity chromatography is a technique used to separate and purify biomolecules based on their specific interactions with ligands attached to a solid matrix. This method allows for the selective isolation of proteins, nucleic acids, or other biomolecules by taking advantage of their unique binding properties, making it essential in various applications, including the isolation of DNA from complex mixtures, the purification of recombinant proteins, and the characterization of proteins in research.
Bacterial artificial chromosomes (BACs): Bacterial artificial chromosomes (BACs) are large DNA constructs used to clone DNA fragments in bacterial cells. BACs can carry large inserts of DNA, typically ranging from 100 to 300 kilobases, which makes them valuable tools in genome mapping and sequencing. Their ability to stably replicate within bacteria allows researchers to maintain and manipulate large segments of genetic material for various applications, including the study of gene function and expression.
Biotechnology regulations: Biotechnology regulations are the legal frameworks and guidelines that govern the research, development, and commercialization of biotechnological products and processes. These regulations are essential to ensure safety, efficacy, and ethical considerations in the manipulation of biological systems, especially when it comes to protein expression systems used for producing therapeutic proteins, enzymes, and other biomolecules.
Eukaryotic expression systems: Eukaryotic expression systems are biological tools used to produce proteins in eukaryotic cells, which have a defined nucleus and complex cellular structures. These systems leverage the cellular machinery of eukaryotes, like yeast, insect, or mammalian cells, to effectively translate, fold, and post-translationally modify proteins, allowing for the production of functional proteins that closely resemble their natural forms. This makes them essential for producing recombinant proteins for research, therapeutic, and industrial applications.
Gene cloning: Gene cloning is the process of making identical copies of a specific segment of DNA, allowing researchers to isolate and study individual genes. This technique is essential for understanding gene function, manipulating genetic material for various applications, and producing proteins or organisms with desired traits. By employing tools like restriction enzymes and DNA ligases, scientists can create recombinant DNA, which serves as a foundation for further applications in biotechnology.
Genetic modification ethics: Genetic modification ethics refers to the moral principles and societal considerations that arise from the manipulation of an organism's genetic material. This includes debates over the safety, consequences, and implications of using techniques like gene editing, which can lead to profound changes in living organisms. The ethics surrounding genetic modification also involves questions about the impact on biodiversity, food security, and the potential for unintended effects on ecosystems and human health.
Gst-tag: The gst-tag, or glutathione S-transferase tag, is a protein domain used in biotechnology for the purification and detection of recombinant proteins. It helps to facilitate the isolation of target proteins by binding them to glutathione-affinity chromatography columns, which takes advantage of the strong interaction between the gst-tag and glutathione. This tag not only enhances protein solubility but also aids in the ease of purification processes within protein expression systems.
Herbert Boyer: Herbert Boyer is a prominent American biochemist known for his pioneering work in recombinant DNA technology and biotechnology. His groundbreaking research in the 1970s laid the foundation for the development of gene cloning and has been instrumental in the creation of genetically modified organisms and therapeutic proteins, impacting various fields including medicine, agriculture, and research.
His-tag: A his-tag is a short sequence of histidine residues, typically six or more, that is added to a protein to facilitate its purification and detection. This tag binds specifically to metal ions like nickel or cobalt, allowing for the easy separation of the tagged protein from other cellular components using affinity chromatography. The his-tag is widely used in various expression systems to simplify the purification process of recombinant proteins.
Industrial enzymes: Industrial enzymes are proteins that catalyze chemical reactions in industrial processes, enhancing efficiency and specificity. They play a crucial role in various sectors including food production, textiles, and biofuels, where they help to reduce energy consumption and minimize waste. Their production often relies on advanced techniques like protein expression systems and is further refined through protein engineering and directed evolution to improve their performance under specific industrial conditions.
Paul Berg: Paul Berg is a pioneering American biochemist known for his groundbreaking work in recombinant DNA technology, which laid the foundation for modern biotechnology. His research led to the development of techniques for gene cloning and the use of plasmids as vectors, greatly influencing how genetic engineering is approached in various fields, including medicine and agriculture.
Plasmids: Plasmids are small, circular, double-stranded DNA molecules that exist independently of chromosomal DNA within a cell. They can carry genes that provide various advantages, such as antibiotic resistance or the ability to produce specific proteins, making them crucial tools in biotechnology and genetic engineering. Their ability to replicate independently allows for the manipulation and transfer of genetic material across different organisms, which is essential for creating gene libraries, expressing proteins, engineering metabolic pathways, transforming cells, and editing genomes.
Post-translational modification: Post-translational modification refers to the chemical changes that proteins undergo after translation, which can alter their function, activity, localization, and stability. These modifications play a crucial role in regulating protein functions and are essential for the proper functioning of proteins within various expression systems, impacting how proteins are utilized in biotechnology and research.
Prokaryotic expression systems: Prokaryotic expression systems are biological systems that utilize prokaryotic organisms, primarily bacteria, to produce proteins by expressing genes. These systems are favored for their simplicity, speed, and cost-effectiveness in producing large quantities of proteins, making them essential tools in biotechnology and research.
Protein refolding: Protein refolding is the process of restoring a denatured or misfolded protein back to its native, functional conformation. This is crucial for proteins synthesized in expression systems that may not fold correctly due to the presence of harsh conditions or other factors. Proper folding is essential for protein functionality, stability, and overall cellular health.
Recombinant DNA Technology: Recombinant DNA technology is a set of methods used to isolate and combine DNA from different sources, allowing scientists to create new genetic combinations that can lead to the production of specific proteins or traits. This technology forms the foundation for various applications in biotechnology, enabling advancements in areas such as medicine, agriculture, and genetic research.
Size-exclusion chromatography: Size-exclusion chromatography (SEC) is a technique used to separate molecules based on their size as they pass through a porous medium. This method is particularly important in the purification and analysis of biomolecules such as proteins and nucleic acids, allowing researchers to isolate specific components by their size, which helps in understanding their structure and function.
Therapeutic proteins: Therapeutic proteins are biologically active proteins designed to treat various medical conditions, including chronic diseases and genetic disorders. They are often produced using advanced biotechnology techniques and can include hormones, antibodies, enzymes, and other proteins that have specific therapeutic effects. The production and optimization of these proteins heavily rely on effective expression systems and innovative engineering methods to enhance their efficacy and stability.