Protein is an important component of all organisms. Each of its molecules consists of one or more polypeptide chains consisting of amino acids. Although the information necessary for life is encoded by DNA or RNA, recombinant proteins perform a wide range of biological functions in organisms, including enzymatic catalysis, protection, support, movement, and regulation. According to their functions in the body, these substances can be divided into different categories, such as antibodies, enzymes, structural component. Given the important functions, such compounds have been extensively studied and widely used.
In the past, the main way to obtain a recombinant protein was its isolation from a natural source, which, as a rule, is inefficient and time-consuming. Recent advances in biological molecular technology have allowed the cloning of DNA encoding a specific set of substances into an expression vector of substances such as bacteria, yeast, insect cells, and mammalian cells.
Simply put, recombinant proteins are translated by exogenous DNA products in living cells. Their receipt usually contains two main stages:
- Cloning a molecule.
- Protein expression.
Currently, the production of such a structure is one of the most powerful methods used in medicine and biology. The composition is widely used in research and biotechnology.
Medical direction
Recombinant proteins provide important treatments for various diseases, such as diabetes, cancer, infectious diseases, hemophilia and anemia. Typical formulations of such substances include antibodies, hormones, interleukins, enzymes, and anticoagulants. There is a growing need for recombinant formulations for therapeutic use. They allow you to expand treatment methods.
genetic engineering recombinant proteins play a key role in the therapeutic drug market. Currently, most therapeutic substances are produced in mammalian cells, since their compositions are capable of producing high-quality substances similar to natural ones. In addition, many approved recombinant therapeutic proteins are produced in Escherichia coli due to their good genetics, rapid growth, and highly productive production. It also has a positive effect when developing drugs based on this substance.
Researching
The production of recombinant proteins is based on different methods. Substances help to find out the basic and fundamental principles of the body. These molecules can be used to identify and locate the substance encoded by a particular gene, and to reveal the function of other genes in various cellular activities, such as cell signaling, metabolism, growth, replication and death, transcription, translation and modification of the compositions considered in this article .
Thus, the observed composition is often used in molecular biology, cell biology, biochemistry, structural and biophysical research, and many other fields of science. Moreover, the preparation of recombinant proteins has international practice.
Such formulations are useful tools in understanding cell-cell interactions. They have been proven effective in several laboratory methods, such as ELISA and immunohistochemistry (IHC). Recombinant proteins can be used to develop enzyme assays. When used in combination with a pair of appropriate antibodies, cells can be used as standards for the application of new technologies.
Biotechnology
Recombinant proteins containing the amino acid sequence are also used in industry, food, agriculture and bioengineering. For example, in animal husbandry, enzymes can be added to food to increase the nutritional value of feed ingredients, reduce costs and waste, maintain animal intestines, improve productivity, and improve the environment.
In addition, lactic acid bacteria (LAB) have been used for a long time for the production of fermented foods, and recently LAB was developed for the expression of recombinant proteins containing the amino acid sequence, which can be widely used, for example, to improve digestion of humans, animals, and nutrition.
However, such substances also have limitations:
- In some cases, the production of recombinant proteins is complex, expensive, and time-consuming.
- Substances produced in cells may not coincide with natural forms. This difference can reduce the effectiveness of therapeutic recombinant proteins and even cause side effects. In addition, this difference may affect the results of experiments.
- The main problem for all recombinant drugs is immunogenicity. All biotechnological preparations may exhibit some form of immunogenicity. It is difficult to predict the safety of new therapeutic proteins.
In general, advances in biotechnology have increased and contributed to the production of recombinant proteins for various applications. Although they still have some disadvantages, substances are important in medicine, research, and biotechnology.
Disease connection
recombinant protein is not harmful to humans. This is only an integral part of the overall molecule when developing a specific drug or nutrient. Many medical studies have shown that the forced expression of the FGFBP3 protein (abbreviated BP3) in a laboratory strain of obese mice showed a significant reduction in their fat mass, despite a genetic predisposition to consume.
The results of such experiments show that the FGFBP3 protein can offer a new therapy for eliminating metabolic syndrome-related disorders such as type 2 diabetes and fatty liver. But since BP3 is a natural protein and not an artificial drug, clinical trials of recombinant human BP3 may begin after a final round of preclinical studies. On, that is, the reasons associated with the safety of conducting such studies. Recombinant protein is not harmful to humans due to its stepwise processing and purification. Changes are taking place at the molecular level.
PD-L2, one of the key players in immunotherapy, who received the 2018 Nobel Prize in Physiology or Medicine. This work, begun by Professor James P. Allison of the USA and Professor Tasuku Honjo of Japan, led to the treatment of cancer, such as melanoma, lung cancer, and others, based on checkpoint immunotherapy. Recently, AMSBIO has added an important new product to its immunotherapy line - PD-L2 / TCR activator - CHO Recombinant cell line.
In experiments to test the concept, researchers from the University of Alabama in Birmingham under the direction of Dr. med. H. Long Zheng, Professor Robert B. Adams and the director of the laboratory medicine department at the pathology department at UAB School of Medicine, highlighted the potential treatment of a rare but fatal disorder blood coagulation, TTP.
The results of this study demonstrate for the first time that transfusion of platelets loaded with rADAMTS13 may be a new and potentially effective therapeutic approach to arterial thrombosis associated with congenital and immuno-mediated TTP.
Recombinant protein is not only a nutrient, but also a drug in the composition of the drug being developed. These are just not many of the areas that are now involved in medicine and related to the study of all its structural elements. As international practice shows, the structure of the substance makes it possible at the molecular level to deal with many serious problems in the human body.
Vaccine development
A recombinant protein is a specific set of molecules that can be modeled. A similar property is used in the development of vaccines. A new vaccination strategy, also known as a special recombinant viral injection, can protect millions of chickens at risk of a serious respiratory illness, researchers at the University of Edinburgh and the Pirbright Institute said. These vaccines use harmless or weak versions of the virus or bacteria to introduce microbes into the cells of the body. In this case, the experts used recombinant viruses with different spike proteins as vaccines to create two versions of a harmless virus. There are many different drugs built on this connection.
Recombinant protein trade names and analogues have the following:
- "Fortelizin".
- "Zaltrap".
- Eilea.
These are mainly anticancer drugs, but there are other areas of treatment associated with this active substance.
According to a new study published in the scientific journal Nature Communications, a new vaccine, also called LASSARAB, designed to protect people from both Lassa fever and rabies, has shown promising results in preclinical studies. Inactivated recombinant vaccine candidate uses attenuated rabies virus.
The research team inserted the Lass virus genetic material into the rabies virus vector so that the vaccine expresses surface proteins in both Lass and rabies cells. These surface compounds elicit an immune response against infectious agents. Then, such a vaccine was inactivated to “kill” the live rabies virus used to make the carrier.
Production Methods
There are several substance production systems. The general method for producing recombinant protein is based on obtaining biological material from synthesis. But there are other ways.
There are currently five major expression systems:
- The expression system of E. Coli.
- Yeast expression system.
- Insect cell expression system.
- Mammalian cell expression system.
- Cell-free protein expression system.
The latter option is particularly suitable for the expression of transmembrane proteins and toxic compounds. In recent years, substances that are difficult to express by conventional intracellular methods have been successfully integrated in vitro cells. In Belarus, the production of recombinant proteins has been widely used. There are a number of state enterprises dealing with this issue.
The cell-free protein synthesis system is a quick and effective method for the synthesis of target substances by adding various substrates and energy compositions necessary for transcription and translation in the enzyme system of cell extracts. In recent years, the advantages of cell-free methods of such types of substances as complex, toxic membrane ones gradually appear, which demonstrates their potential application in the biopharmaceutical field.
Cell-free technology can easily and in a controlled manner add a variety of non-naturally occurring amino acids to achieve complex modification processes that are difficult to solve after conventional recombinant expression. Such methods are of high value for use and the potential for drug delivery and vaccine development using virus-like particles. A large number of membrane proteins have been successfully expressed in free cells.
Expression of compounds
Recombinant protein CFP10-ESAT 6 is produced and used to create vaccines. Such a tuberculosis allergen can enhance immunity and develop antibodies. In general, molecular studies include the study of any aspect of a protein, such as structure, function, modifications, localization, or interactions. To investigate how specific substances regulate internal processes, researchers usually require tools to produce functional compounds of interest and benefit.
Given the size and complexity of proteins, chemical synthesis is not a viable option for this endeavor. Instead, living cells and their cellular mechanisms are usually used as factories to create and construct substances based on the provided genetic patterns. The recombinant protein expression system further develops the necessary structure to create the drug. Next, the necessary material is selected for a different category of drugs.
Unlike proteins, DNA is easy to construct synthetically or in vitro using well-established recombinant techniques. Therefore, DNA matrices of specific genes, with or without added reporter or affinity tag sequences, can be designed as templates for the expression of the monitored substance. Such formulations derived from such DNA templates are called recombinant proteins.
Traditional strategies for expressing a substance include transfecting cells with a DNA vector that contains a template and then culturing the cells so that they transcribe and translate the desired protein. Typically, the cells are then lysed to extract the expressed composition for subsequent purification. The recombinant protein CFP10-ESAT6 is processed in this way and goes through a system of purification from the possible formation of toxins. Only after that he enters the vaccine for synthesis.
Both prokaryotic and eukaryotic in vivo molecular expression systems are widely used. The choice of system depends on the type of protein, the requirements for functional activity and the desired yield. These expression systems include mammals, insects, yeast, bacteria, algae, and cells. Each system has its own advantages and problems, and the choice of the right system for a specific application is important for the successful expression of the substance examined in the article.
Mammalian expression
The use of recombinant proteins allows the development of vaccines and drugs of various levels. For this, this method of obtaining the substance can be used. Mammalian expression systems can be used to produce proteins from the animal world that have the most native structure and activity due to their physiologically relevant environment. This leads to high levels of post-translational processing and functional activity. Mammalian expression systems can be used to produce antibodies, complex proteins and compounds for use in cell-based functional assays. However, these advantages are combined with more stringent cultural conditions.
Mammalian expression systems can be used to produce proteins temporarily or through stable cell lines where the expression construct is integrated into the host genome. While such systems can be used in several experiments, temporary production can generate large quantities of matter in one to two weeks. The biotechnology of recombinant proteins of this type is in high demand.
These transient, highly productive mammalian expression systems utilize suspension cultures and can yield grams per liter. In addition, these proteins have more native folding and post-translational modifications, such as glycosylation, compared to other expression systems.
Insect expression
Methods for producing recombinant protein are not limited to mammals. There are more productive methods in terms of production costs, although the yield of a substance per 1 liter of processed liquid is much lower.
Insect cells can be used to express high-level protein with modifications similar to mammalian systems. There are several systems that can be used to produce recombinant baculovirus, which can then be used to extract the substance of interest in insect cells.
Expression of recombinant proteins can be easily expanded and adapted to a high density suspension culture for large-scale production of a compound of molecules. They are more functionally similar to the native composition of the substance of mammals. Although the yield can be up to 500 mg / l, the production of recombinant baculovirus can take a lot of time and the culture conditions are more complex than prokaryotic systems. .
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Although they are not resistant to large-scale production, cell-free systems or in vitro protein expression systems (IVT) have several advantages over traditional in vivo systems.
Cell-free expression allows the rapid synthesis of recombinant formulations without the involvement of cell culture. Cell-free systems allow the labeling of proteins with modified amino acids, as well as the expression of compounds that undergo rapid proteolytic degradation by intracellular proteases. In addition, using the cell-free method, it is easier to simultaneously express many different proteins (for example, to test protein mutations by expressing on a small scale from many different recombinant DNA matrices). In this representative experiment, the IVT system was used to express human caspase-3 protein.
Conclusions and prospects for the future
The production of recombinant protein can now be seen as a mature discipline. This is the result of numerous gradual improvements in purification and analysis. Currently, drug discovery programs rarely stop because of the inability to produce the target protein. Parallel processes for the expression, purification and analysis of several recombinant substances are currently well known in many laboratories around the world.
Protein complexes and the growing success in creating solubilized membrane structures will require more changes to keep up with demand. The emergence of effective contract research organizations for a more regular supply of proteins will redistribute the resources of science to solve these new problems.
Additionally, parallel workflows should allow the creation of complete libraries of the substance being monitored to enable the identification of new targets and advanced screening, along with traditional small molecule drug discovery projects.