There is no doubt that modern technologies greatly simplify human life, especially if it concerns computers and intelligent systems. Being a part of a developed digital society, the consumer integrates with existing technical solutions every day and, therefore, hardly perceives them as something fantastic. In biological technologies, the situation seems to be the opposite since the methods being developed do not seem to be a part of daily human life. In reality, thanks to the work of biotechnologists, millions of people around the world have access to the vital insulin hormone synthesized by genetic engineering techniques. Such grand public importance of works performed on a microscopic level in a microbiological laboratory determines the interest of the author in discussing this synthesis. This work summarizes the technology for obtaining genetically modified insulin by manipulating the E. coli genome.
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Discussion of relevance of the problem
The description of the mechanism of artificial hormone production should be preceded by a discussion of the relevance of this issue. A serious problem for people with both types of diabetes is the lack of naturally produced insulin. It is known that the body of patients with this diagnosis is not able to independently synthesize the biologically active substance or produce it in insufficient concentrations (Katsarou et al. 2). This, in turn, leads to delays in the absorption of glucose, resulting in persistent hyperglycemia. Elflein estimates that about ten percent of the adult population suffers from this disease, which necessitates the development of therapy (Elflein). Diabetes, being an almost incurable disease, requires a permanent source of insulin obtained synthetically.
Indeed, animal insulin was a good solution for this problem, but its quantity was not enough in the conditions of the spreading epidemic of diabetes. Moreover, animal insulin production was associated with obvious economic and technological difficulties due to the high cost, challenges in the extraction, storage, and transportation of raw materials (Cho et al. 550). Therefore, back in 1978, biotechnologies were able to obtain the first artificial insulin created using the technology of translation of prokaryotes, E. coli (Vecchio et al. 6). As an example of a successful scientific application process, this solution is still popular in research laboratories around the world, where insulin is produced for patients in need.
The principal method of this process boils down to manipulating the genome of E. coli, into which the necessary gene is injected. The technology begins with the fact that the Beta cells — who are the producers of the hormone — localized in the human pancreas are treated with alkaline phosphatase to extract a pure gene of interest, as shown in Figure 1. It is known that this nucleotide sequence is located on the short shoulder of the 11th chromosome (“INS Insulin”). In addition, the reference gene used does not necessarily have to be enzymatically isolated from the host body but can be chemically synthesized since the specific sequence of nucleotides has been deciphered before. The next step is to remove a DNA ring molecule called a plasmid from the vector organism: usually a bacterium. At this stage, it is important to use cutting-edge enzymes, restriction endonucleases, which will split the phosphodiester bonds inside the plasmid into the same restriction sites as in the human gene: in this way, laboratory technicians obtain a linear form of the ring molecule (“Production of Recombinant Protein”). Then, the isolated human gene is mixed with the torn plasmid, resulting in recombinant bacterial DNA that is capable of insulin biosynthesis. The use of ligases makes it possible to cross-link the sugar-phosphate skeletons of the two sticky ends of DNA chains to create a complete double helix structure.
Subsequently, the obtained DNA chain must be placed in a vector cell capable not only of accepting foreign genetic material but also of synthesizing a hormone. This process involves several complexities since the vast majority of cells traditionally reject the inserted material: they die or split the insertion gene. That is why the placement of plasmid in the cell can take place in several ways, among which, as a rule, the transformation prevails. As a result that, competent bacteria absorb the material that was offered to them. Selected cells with built-in recombinant material are cultivated on the nutrient medium, producing insulin activity. The resulting biologically active substances are purified by chemical-enzymatic technologies and fed into pharmaceutical companies.
There is no doubt that this biotechnology technique is of high importance for medical organizations and patients. However, the general scheme of introducing any gene into vector plasmid to produce recombinant human proteins can be scaled up for alternative research as well. In particular, utilizing genetic engineering methods, such essential molecules of the body as interferons, somatotropin, and erythropoietin, can be obtained. Further development of this scheme can be reduced to the search for more effective producers, showing a higher survival rate combined with the expansion of the spectrum of genes introduced into the prokaryotic cell.
Summing up, it should be noted that biotechnological methods are focused on solving problems that are important for society. Diabetes mellitus is a very common diagnosis among patients, and about one in ten adults suffers from this disease. The urgency of finding a biotechnological solution to public health was due to the need for cheap and high-quality insulin synthesis in laboratory conditions. This was done back in the second half of the last century, and since then, the developed scheme of hormone production has not changed much. Thus, artificial insulin synthesis requires the injection of an isolated human gene in a plasmid vector isolated from the prokaryotic cell. The recombinant DNA obtained is transformed into a vector cell, for example, E. coli, after which the cells are cultivated.
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It was shown that this technology has a high potential for future technological research. The range of work can be extended to the search for new vector organisms capable of better survival and introducing new gene agents into plasmids. In the future, this means that with the help of genetic engineering, it will be conceivable to obtain any recombinant proteins for the needs of patients. In many respects, the probability of such works is due to the relative cheapness of the procedure in comparison with analogs, simplicity of classical synthesis schemes, and unlimited application. It is interesting to note that this hormone is a vegan product, and therefore suitable for more patients. On the other hand, the cost of biotechnological synthesis is still quite high, and genetically modified insulin requires a thorough biological safety test.
Cho, Bumrae, et al. “Production of Genetically Modified Pigs Expressing Human Insulin and C-Peptide as a Source of Islets for Xenotransplantation.” Transgenic Research, vol. 28, no. 5-6, 2019, pp. 549-559.
Elflein, John. “Diabetes – Statistics & Facts.” Statista. 2019, Web.
“Genetic Modification and Cloning.” BBC, n.d., Web.
“INS Insulin [ Homo sapiens (Human) ].” NCBI, n.d., Web.
Katsarou, Anastasia, et al. “Type 1 Diabetes Mellitus.” Nature Reviews Disease Primers, vol. 3, no. 1, 2017, pp. 1-17.
“Production of Recombinant Protein.” CusaBio. 2018, Web.
Vecchio, Ignazio, et al. “The Discovery of Insulin: an Important Milestone in the History of Medicine.” Frontiers in Endocrinology, vol. 9, 2018, 1-8.