Researchers can now express heterologous proteins in various biological systems due to advancements in cloning, genomics, and numerous molecular biology techniques. Researchers have a wide range of powerful downstream applications available due to the ability to express recombinant proteins, which they use to further their research studies. Proteins expressed on a small scale are primarily used for studying and verifying protein functions.
In contrast, those produced on a large scale are significant in studying enzymes, antibodies, and vaccine production, hence the importance of determining optimal cell growth and protein expression conditions for small- and large-scale systems. Whether a prokaryotic or eukaryotic expression system is required for post-translational modifications, the cell type will significantly impact the tools and reagents needed for optimum protein expression. This article will look at protein expression and purification services in detail.
What is protein expression?
Protein expression is the process by which proteins are produced, modified, and regulated in living organisms. Custom protein expression can be synthesized through laboratory techniques, while recombinant protein production is achieved through cellular machinery. Additionally, scientists and researchers have been developing new protein expression techniques to help simplify the process. Several scientists and laboratories offer quality protein production services.
What is a recombinant protein?
To understand how custom protein is synthesized, we must ask ourselves what recombinant proteins are. Recombinant proteins are those that have been changed for maximum protein expression efficiency or altered to assess protein function and are encoded within a protein-expressing plasmid.
Researchers can use the capacity to add, remove, or change the protein-encoding sequence by a single nucleotide to examine a wide range of fundamental scientific problems and elucidate the protein's function in both healthy and diseased tissues. Recombinant protein expression technology has far-reaching implications beyond basic research and is critical for creating life-saving medicines and vaccines.
Protein translation and transcription
Transcription is an essential process in the collection of molecular tools and the creation of pathways for protein expression. Transcription manifests in three steps**: initiation, elongation,** and termination. Transcription starts when the double-stranded DNA is unwound to enable the binding of RNA polymerase. In eukaryotes, the transcription process is regulated through repressors and activators. However, in prokaryotes, there is less specialized regulation of RNA.
Similarly, the translation process requires macromolecules such as ribosomes and tRNA, and micromolecules such as amino acids, GTP, and ATP. Translation and transcription of protein are essential processes that help in custom protein expression. After the translation process, polypeptides are modified in different ways to complete their structure and designate their location. The modifications that take place after translation include polypeptide folding, modification of amino acids, disulfide bridge formation, and glycosylation.
Protein expression host systems
For protein expression, a host system must be selected. There are different host systems, such as bacterial host systems and mammalian cells. Bacteria are the workhorse organisms for manufacturing recombinant proteins, catalyzed by rapid growth kinetics and plasmid conversions in E. coli. The 30S and 50S ribosomal subunits of the 70S bacterial ribosome are required for bacterial protein expression. Plasmids harboring antibiotic-resistance genes are employed as a selection tool to detect and isolate bacteria that have taken up plasmids containing the protein-encoding sequence of interest, preventing the growth of plasmid-free cells.
While additional genetic sequencing is frequently required to validate the presence of your gene sequence, plasmid-free bacteria are commonly removed using antibiotics that impede bacterial protein synthesis. The bacterial expression system has a short doubling time, making it a convenient host cell for expression. Insect, yeast, and mammalian cell lines are also commonly employed for protein expression. Eukaryotic cell lines have different molecular machinery for generating post-translational modifications (such as glycosylation).
These modifications are often required for protein functionality and proper downstream analysis. However, it is important to note that some expression systems, such as bacteria, may contain toxic pyrogens, so the synthesized protein should be effectively tested. Therefore, a suitable purification method must be applied during the protein production process. E coli expression systems are the most preferred host systems in custom protein expression because they provide a high protein yield.
Protein expression vectors
Expression vectors, also known as plasmids, are circular DNA sequences often utilized by scientists to host the gene encoding their desired protein. Plasmids encoding the desired gene are transformed or transfected into cells to overexpress a protein. Plasmids contain a multiple cloning site (MCS), antibiotic resistance genes for clone selection, unique tags for protein expression and purification, and vital promoter regions to drive protein expression. Other valuable elements facilitate cloning, selection, and protein purification.
Protein expression vectors come in various shapes and sizes, with many of these components interchangeable depending on the needs of the application and the cell type utilized. The purpose of protein expression vectors is to act as promoter regions and enhancers, ensuring effective transcription of the gene carried in the vector, and to provide efficient production of protein. Protein expression through these vectors is achieved by producing considerable amounts of RNA, which is then translated into protein.
Protein expression conditions
Proteins can only be expressed under specific conditions. Recombinant protein is often generated using the chemical isopropyl-β-D-1-thiogalactopyranoside (IPTG) or through the alteration of carbon sources found in E. coli cells. In both methods, the cells are grown at high densities, especially when exposed to conducive conditions such as baffled shake flasks. However, the expression should be induced in the mid-to-late log phase regardless of the cell density to ensure maximal yield. This helps avoid challenges that arise during the stationary phase, such as the generation of proteases.
Most expressed proteins thrive at moderate temperatures and remain soluble at 15–25°C. These temperature conditions allow the newly produced proteins to fold correctly. Therefore, temperature regulation is crucial during induction and the entire production process. To understand the details of the protein production process, one can learn more through laboratories that offer professional protein expression services.
Antibody synthesis
Antibodies are immune proteins that aid in the fight against viruses and bacteria. Antibody-based medicines can help in the treatment of infections and other diseases. For antibody production, a laboratory or farm animal is injected with a specific antigen, which leads to the production of antibodies in the serum. The produced antibodies are then retrieved from the animal. Monoclonal antibodies are produced in hybridoma cell lines through the fusion of antibody-secreting spleen cells from the immunized animal with myeloma cells. Antibody sequencing services help identify the amino acid sequence of any antibody. Monoclonal antibody sequencing is done through hybridoma cell line sequencing and clonal B cell sequencing. In addition, antibody specificity is essential in identifying the "goodness of fit" between a paratope and an epitope.
Several biochemistry laboratories offer antibody production services. Antibodies play an essential role in protein research, and they are major components of the immune system involved in fighting germs. Custom antibody production services have advanced due to technological improvements. In addition, custom antibody production guides are now more accessible, allowing researchers to follow them easily.
Peptides are essential components in the production of custom antibodies. Peptide synthesis was historically one of the most challenging and tiring tasks with low yields. However, with advancements in technology, peptide production is more straightforward and gives higher output in less time. Peptide synthesis can be classified as the formation of peptide bonds between two amino acids. Peptides are usually flexible chains of amino acids.
With modern technology and research knowledge in the chemistry field, it is possible to produce peptides with different designs and unique biological responses. There are various methods of peptide production, such as solid-phase peptide synthesis (SPPS). Just like proteins, peptides must undergo a purification process after production. Purification helps to remove any side products that the reactions may have formed during the production process, such as isomers.
Gene production
Genes are responsible for encoding proteins. Therefore, gene expression is a crucial factor in protein expression, as it determines the functions of a particular cell. Foreign gene expression is affected by a variety of factors, such as transcriptional regulators, ribosome binding sites, the location of the cloned protein within the host cell, the metabolic rate of the cell, the stability of the codons utilized in the foreign gene, and the number of foreign genes expressed. The amount of particular proteins synthesized in any cell depends on the balance between the synthetic and degradative biochemical pathways of the protein.
Therefore, there should be regulation of gene expression throughout the entire process of protein synthesis. Gene expression is regulated within the nucleus, especially at the transcriptional level in eukaryotes. Notably, only a specific number of genes are expressed in cells at a particular time. Gene expression is usually affected by environmental changes and requires regulatory mechanisms. The availability of nutrients affects prokaryotic gene expression, allowing bacterial organisms to change their transcription patterns in response to environmental conditions effectively.
Protein purification
Protein purification is a set of procedures for isolating a specific protein from a complex mixture. Purifying the protein of interest is critical for determining its function, interactions, and structure. Purification separates the desired protein from other components, dividing the target protein from unwanted proteins. The most time-consuming aspect of protein purification is usually separating one protein from all others. The most common properties utilized in the separation process are biological and physicochemical features, binding affinity, and differences in protein size. "Protein isolate" is the term for the pure outcome. Protein purification is a significant stage in custom protein expression that can be done preparatively or analytically.
There are four fundamental protein purification steps:
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Cell lysis
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Protein binding to a matrix
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Washing
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Elution
Methods of protein purification
Extraction
Extraction is a purification step that involves bringing the protein into solution from the cells by breaking the tissues or cells of the host system. Several methods are used, such as sonication, recurrent freezing and thawing, and homogenization. The extraction method applied depends on the fragility of the protein and the host cells. Soluble proteins remain in the solvent and can be separated from the cell membrane through centrifugation.
Differential solubilization and precipitation methods are suitable for large-scale protein purification. In this method, protein is precipitated using ammonium sulfate. This is achieved by adding increasing concentrations of ammonium sulfate and retrieving the various fractions of the precipitated protein. This strategy is beneficial as one can purify large volumes of protein at a lower expense.
Ultracentrifugation
In this method, centrifugal force is used to separate mixtures of particles of varied weights or densities suspended in a liquid. A tube with a protein mixture is spun at high speeds, where the angular momentum yields an outward force on every particle proportional to its mass. Due to this force, the particles move through the liquid at different rates.
Chromatography
Chromatography is a protein purification method that often involves more than one chromatographic step. Flowing a solution containing the protein through a column filled with various materials is the basic technique. Different proteins interact differently with the column material; they can be distinguished by the time it takes to pass through the column or the conditions under which the protein elutes. Different chromatography methods are used in protein purification: Size-Exclusion Chromatography, Ion-Exchange Chromatography, Affinity Chromatography, Metal-Binding (IMAC), and Immunoaffinity Chromatography.
Purpose and importance of protein purification
Purification of proteins is essential for determining their function, structure, and interactions. The primary purpose of protein purification can be divided into:
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Preparative purification: This aims to create large quantities of purified proteins for later usage. Several preparative procedures are used to remove byproducts, such as host cell proteins, which could pose a health risk to patients.
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Analytical purification: This yields a small amount of protein for research or analytical objectives, such as protein identification, quantification, structural analysis, post-translational modification studies, and function studies. The first proteins to be purified to the point that they could be crystallized were pepsin and urease.
Final thoughts
Protein expression and purification are topics that have attracted significant attention in the biochemistry field. Protein expression can be defined as the process by which living cells produce and modify proteins. Protein production is also achieved through laboratory technology. There is another method of protein expression known as chemical synthesis.
This method is significant for producing proteins with unnatural amino acids, proteins that are toxic to biological systems, or proteins labeled at specific sites. Chemical protein synthesis yields high-purity protein but is only convenient for small proteins and peptides. This method can prove to be expensive for larger quantities of protein.