The production of recombinant proteins using microbial systems has helped rapidly revolutionize biochemistry. We are past the era where large amounts of plant and animal tissues or large volumes of biological fluids were required to purify small amounts of a given protein. Anytime you carry out a project that requires purified proteins, you should opt for recombinant proteins, which are easy to express and purify. The ability to efficiently produce and purify recombinant proteins in large quantities ensures easy biochemical characterization, commercial product development, and industrial use.
The steps required for expressing recombinant proteins are simple. Additionally, with improved technology, the process is faster and easier than ever. Nevertheless, there are possible impurities that are likely to accumulate during the process, so your protein must undergo thorough purification.
This article will explore the various benefits of recombinant protein purification.
What are Recombinant Proteins?
Before proceeding, let’s begin by gaining a quick idea of what recombinant proteins are. Essentially, recombinant protein production happens through the transfection of foreign genes into a host cell. There are various recombinant protein uses, such as producing pharmaceutical products, antibodies, and recombinant enzymes for the treatment of diseases, as well as protein-based polymers for drug delivery and protein scaffolds for tissue engineering, among many other applications. Researchers also use recombinant proteins in their daily projects.
The Emergence of Recombinant Protein
Peter Lobban first proposed the idea of recombinant proteins. He was the first to express and replicate recombinant DNA in 1973. Recombinant DNA is produced through the fusion of sequences that may not naturally occur in an organism. Nevertheless, you can change and re-insert modified DNA because most organisms have similar DNA structures.
The altered DNA is then introduced to the host cell, after which it is either replicated without any expression or transcribed and translated, leading to the creation of a recombinant protein. In most cases, additional sequences, such as translation initiation signals or promoters, may be needed to facilitate the expression of the recombinant DNA. The expression of recombinant proteins has, however, evolved and improved tremendously. Recombinant protein technology is continuing to improve as more efficient methods keep emerging.
Expression of Recombinant Protein
The methods used to produce recombinant proteins involve transfecting specific cells with a DNA vector containing a template of recombinant DNA. The cells containing the template are then cultured to facilitate the transcription and translation of the desired protein.
The cells can then be broken or lysed to release the created proteins, which are later purified. The systems used in expressing recombinant DNA include both eukaryotic and prokaryotic systems. The choice of system is determined by the type of protein you need, its functional activity, and the yield required.
Common Host Systems Used in Recombinant Protein Expression
There are several recombinant protein expression systems commonly used:
1. Mammalian Systems
Mammalian systems are used for various purposes, such as performing structural analysis, studying protein interactions, custom antibody production, functional assays, and the production of viruses. The main advantage of using this system is the ability to achieve stable protein production, and the fact that the system is also amenable to transient expression.
Additionally, the system can be used for the rapid production of large quantities of proteins. Nevertheless, there are some disadvantages associated with the use of mammalian systems. For example, cell culture conditions are more demanding, and there is often a need for large culture volumes (e.g., liters) to achieve high yields when using suspension cultures.
2. Insect System
The insect system is commonly used for expressing protein complexes and intracellular proteins, performing structural analysis, producing viruses, and performing functional assays, among other applications. The expression process in the insect system is similar to the process used in mammalian systems. Nevertheless, the cell culture conditions may be more demanding in an insect system than in prokaryotic cell culturing. Additionally, the production of recombinant vectors may take longer when using an insect system.
3. Yeast System
Like mammalian and insect systems, yeast systems are also used to perform functional assays and structural analysis. Additionally, they are used to analyze generated antibodies and study protein interactions. A yeast system can be applied in processing eukaryotic proteins through fermentation. The system has simple media requirements; nevertheless, to produce high-yield proteins, fermentation is essential, and growth conditions must be optimized.
4. Bacterial System
Bacterial systems are the most commonly used hosts due to their low cost, scalability, and simple media requirements. They can also be used in functional and structural assay analysis. The most commonly used bacterial host is E. coli because it has fast growth kinetics, high-density cultures can be achieved quickly, and the manipulation of recombinant DNA is easy and fast. However, this system may not express some mammalian proteins efficiently.
Example of Recombinant Proteins
- Human insulin, used in the treatment of diabetes
- Human growth factors are used to treat hormone deficiencies
- Factor VIII, which is commonly used to treat hemophilia
- Therapeutic monoclonal antibodies are used in treating cancer and viral infections
- Research reagents, including inhibitors, proteins for ELISA and fluorescence assays, protein-folding studies, and small molecules.
Before the invention of molecular biology and recombinant protein expression technology, these proteins were obtained directly from animal sources. The advent of this technology led to the development of entirely artificial recombinant proteins, such as recombinant antibodies for diagnostic and therapeutic applications and proteins for vaccine production.
Protein Purification
The production of recombinant proteins, primarily through bacterial hosts and vectors, is a well-established and well-defined technology. Nevertheless, isolating them into an active form remains a significant challenge. Therefore, the purification of recombinant proteins is a vital process in biological research. To study a protein's particular structure and function, you must use a pure recombinant protein. This means you must isolate and purify it before use. Protein purification, therefore, is a process that involves separating the target protein from a concentrated mixture of other unwanted tissues, cells, or proteins.
Protein purification technology primarily uses similarities and differences between various recombinant proteins. For example, non-proteinaceous materials such as tissues and cells can be eliminated based on common characteristics. On the other hand, the target protein can be isolated based on unique physical or chemical differences from other proteins.
Protein tags are vital and convenient tools that help streamline protein purification, enhancing the solubility of the required recombinant protein and providing an easy method to track proteins during the recombinant protein expression and purification process. Further, protein tags are essential for tracking proteins and processes using microscopic or indirect methods such as immunoprecipitation, Western blotting, or immunostaining.
Common Purification Methods Used for Recombinant Proteins
1. Extraction
This purification technique isolates the protein by disrupting cells or tissues from the host systems. If the organism is the source of the protein, the first vital step is ensuring the cells are lysed where the proteins are located. You can use various methods such as repeated freezing and thawing, homogenization via high grinding pressure, sonication, or permeabilization using enzymes or detergents. Additionally, the extraction method releases proteases during cell lysis, which can lead to the digestion of the target protein in the solution.
If there is a reaction between the protein and proteases, you should expedite the purification and ensure the extract is kept cool to slow the process of digestion. Alternatively, you can use protease inhibitors. The extraction method used in the purification process depends on the host cells and protein fragility. Soluble proteins can remain in the supernatant and be separated from the cell membrane via centrifugation.
2. Chromatography
One of the most popular purifying techniques is chromatography. This is a result of its many benefits compared to other purifying techniques. For instance, the high-resolution efficiency of chromatography enables the isolation of complex mixtures. Also, chromatography is a convenient method for trapping target molecules even in a highly diluted solution.
The main idea behind the chromatography technique is to separate large recombinant protein pools into smaller fractions that contain a high concentration of the target protein. The method uses specific and essential equipment and adopts a series of steps. For example, the protein solution flows through columns packed with stationary phase materials as the first important step in this procedure. The column materials allow numerous types of proteins to interact in various ways based on their physical and chemical properties.
Various chromatography strategies are applied in recombinant protein purification, including: size-exclusion chromatography (gel filtration), affinity chromatography, and immunoaffinity chromatography.
Precipitation and Differential Solubilisation
Differential solubilization and precipitation procedures work best for removing significant amounts of contaminating proteins. One of the essential reagents utilized in this approach is ammonium sulfate. It helps precipitate proteins by the addition of varying concentrations of ammonium sulfate, allowing for the collection of different precipitated protein fractions.
Initially, recombinant proteins may undergo the precipitation process to ensure the target proteins are concentrated before moving on to additional processes with specified purification columns. The recombinant protein can also precipitate naturally as an inclusion body due to a variety of variables in the expression host system. Precipitation and differential solubilization are among the most affordable and cost-effective methods for recombinant protein purification and are ideal for purifying large quantities of proteins.
Ultracentrifugation
This purifying technique separates a mixture of molecules with varying densities and masses using centrifugal force. As the sample is spun at high speeds, the acceleration affects every particle in the protein mixture, causing them to exert an outward pressure proportional to their mass. The targeted particles are then sedimented through the liquid by this centrifugal force.
The particles flow through the fluid against the buoyant force applied to them. Compared to less massive molecules, larger and denser particles will move outward faster when you spin the sample in a centrifuge.
Gel Filtration
Gel filtration is a convenient and accessible purification method for separating components based on size. It is a type of chromatography. The stationary phase is first packed into columns to facilitate an easy and fast purification process. This setup results in what is known as a "packed bed." Because particles and molecules have different physical and chemical stabilities, the media must be composed of a porous matrix. The packed bed is equilibrated with a buffer that fills the pores of the matrix and the remaining spaces between particles.
Benefits of Recombinant Protein Purification
- High-Quality Protein: The purification process ensures that you obtain high-quality recombinant proteins free from contaminants or unwanted materials such as host cell tissues or debris. This means that the produced recombinant proteins will yield more reliable outcomes when used in research, medicine, and vaccine production.
- Isolation of Specific Proteins: Initially, produced proteins may contain unwanted proteins from the host system. Purification ensures you isolate the specific target protein by separating it from other cellular proteins. With modern purification methods, it is now possible to obtain a specific pure protein quickly.
- Support for Protein Characterization: Protein Characterization is a process that uses experimental methods to identify and analyze proteins. It helps in defining the structure and interactions of the target protein. Protein purification is a vital prerequisite for the characterization process.
- Assessment of Biological Activity: Through recombinant protein purification, it is possible to assess and establish various biochemical properties, such as signaling capacity and enzymatic activities, that reside in unique proteins.
- Production of Essential Reagents: Pure recombinant proteins are valuable in producing biochemical reagents such as hormones, growth factors, DNA polymerases, proteases, ligases, antibodies, and phosphatases.
- Enzymological Study: Purified proteins enable the study of their enzymology and affinity for specific substrates. Such knowledge provides insight into how various biological molecules work as catalysts in metabolic processes.
- Improved Assay Performance: Purified recombinant proteins make the use of ELISA kits and fluorescence assays more accurate and easier to perform.
Wrapping Up
Recombinant protein purification is a cornerstone of the biochemistry field. Due to continuous and thorough research, recombinant protein expression and purification processes have evolved significantly, enabling the production of high-yield, high-quality proteins. Additionally, the considerable improvement of various purification methods has ensured the consistent yield of pure and specific proteins.
If you lack the necessary equipment, knowledge, or skills, you can seek affordable recombinant protein expression and purification services from a professional laboratory. Before embarking on any research project that requires recombinant proteins, ensure that you can access pure, high-quality proteins to achieve the best results.