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Escherichia Coli in Recombinant Human Insulin Production

Escherichia coli In Recombinant Human Insulin Production
Faris Abuzahra

Abstract
Insulin is the hormone our pancreas produces to allow our cells to use and store glucose we obtain from consuming food. Without insulin our bodies would not be able to control blood sugar levels, making it a necessary hormone for us to survive. People who are diabetic either do not produce insulin or are resistant to it for various reasons. As a result of this they need insulin injections to allow their bodies to process glucose. The production of recombinant human insulin with Escherichia coli has allowed insulin to become more available to patients around the world. In this review, the detailed steps of producing recombinant human insulin using the K12 strain of E. coli, along with why E. coli is the preferred organism for insulin product. This review will also touch on the significance of recombinant insulin.
Introduction
The demand for insulin is on the rise worldwide as the increasing rate of diabetes shows no signs of slowing down. According to the CDC more than a 100 million U.S. adults have been diagnosed as diabetics or prediabetics [1]. The insulin hormone is produced by the pancreas to regulate the sugar levels in blood. In the 1930’s through 1970’s diabetics used insulin that was harvested from animals such as pigs and cows which could cause many allergic reactions in patients. It wasn’t until 1982 when the first recombinant human insulin derived from Escherichia coli was available to diabetic patients [2]. This was done by inserting the human insulin gene the plasmid of the E. coli bacteria which would translate into insulin. This breakthrough allow insulin to become readily available to patients at a more affordable price. The purpose of this review is to explore how Escherichia coli is used to produce recombinant human insulin and why it is the preferred organism for insulin production.
Why E. coli is the best for insulin production
Escherichia coli is the preferred organism for insulin production for many reasons. E. coli has the fastest reproduction rate which under the right conditions can double its numbers every 20-30 minutes. It is also resistant to antibiotics such as ampicillin and tetracycline which allows insulin manufactures to easily inhibit the growth of unwanted microbes when it is fermented on a large scale. E. coli is easy to handle which makes it very cost efficient to maintain. E. coli also produces the highest yields of insulin comparted to other organisms used for its production. All of this makes the production of insulin using E. coli the most profitable for manufactures [3].
Before E. coli was used in the production of recombinant human insulin diabetic patients relied on insulin that was harvested from the pancreases of pigs and cows. Although this insulin worked to help keep the patient’s blood sugar at a normal level the insulin molecules from pigs and cows differed slightly at the insulin receptor binding site. The B-chain in human insulin had a threonine amino acid at the C-terminal, while in pig insulin there was an alanine amino acid. Insulin derived from cow pancreases differed a bit more with having three substitutions with the B-chain having an alanine amino acid on the C-terminal. On the A-chain a valine amino acid is at the A10 position and alanine on the A8 position, unlike human insulin which has isoleucine on the A10 position and threonine on the A8 position [4]. The production cost of insulin derived from pigs and cows was extremely expensive. For example, it would take over two tons of pig pancreases just to produce 8 ounces of insulin. This resulted in the cost of insulin being high and not every diabetic patient could afford to purchase. Another organism used in insulin production is the yeast strain, Saccharomyces cerevisiae [3].Like E. coli, S. cerevisiae is used in the production of recombinant human insulin. The methods used are similar with having a human insulin gene inserted into the plasmid of the S. cerevisiae cell. The down side to using S. cerevisiae is that the productivity rate of recombinant insulin is musch lower when compared to that of E. coli [5]. The productivity rate for E. coli is ~1085 (mg/1 h) at 80 (g/l) of biomass [6]. The productivity rate for S. cerevisiae is ~1.04 (mg/1h) at 5 (g/l) of biomass [7].
The production process of insulin using E. coli
Recombinant human insulin production using Escherichia coli begins with taking the insulin secreting cell from the human pancreas. From that cell the mRNA transcript is taken out to isolate the insulin human gene. This will be done for both the A-chain protein and B-chain protein insulin forming genes that will be combined near the end of the production to form the complete insulin molecule. The enzyme reverse transcriptase is attached to the mRNA which creates a single strand of cDNA. The cDNA is then polymerized by DNA polymerase to form a double strand of DNA. That double stranded DNA is then multiplied by the polymerase chain reaction (PCR) which rapidly makes many copies of the DNA. At this point the DNA strand needs to be place in the plasmid of the E. coli K12 cell [4]. The E. coli plasmid has two antibiotic resistant genes one for tetracycline and the other is an ampicillin resistant gene. The restriction enzyme cutting point is in the middle of the tetracycline resistant gene which is where the plasmid opens to allow the human insulin gene to be inserted. The gaps between the insulin gene and the rest of the plasmid are sealed with DNA ligase to form a complete recombinant plasmid. Since the restriction enzyme cutting point is in the middle of the tetracycline resistant gene once the plasmid is cut and the insulin gene is inserted the recombinant plasmid is no longer tetracycline resistant [3].
The next step would be to insert the plasmids back into the E. coli cells. This is done by placing the cells into calcium chloride to make the cell membranes permeable and the plasmids are added to the mixture. To allow the cells to uptake the plasmids they are either put through heat shock or electroporation. There are four possible outcomes after the E. coli cells uptake the plasmids. One being cells that took the plasmids without the human insulin gene, and cells that took up no plasmids at all, cells that took up insulin genes without plasmids, and cells that took up the desired recombinant plasmids. To identify which E. coli cells have taken up the recombinant plasmids manufactures us antibiotic resistance to distinguish between the four possible outcomes by adding ampicillin and tetracycline. The cells that have a plasmid without the insulin gene would be resistant to both antibiotics. The cells that did not uptake any plasmids are sensitive to both antibiotics. The cells that have to recombinant plasmids would be resistant from ampicillin but sensitive to tetracycline because the restriction enzyme cutting point was in the middle of the tetracycline resistant gene. Figure 1 gives a visual representation of how the human insulin gene is inserted into the plasmid which is then inserted into the E. coli cell [8].

Figure 1. The Process of Recombinant Insulin Production. This figure shows a brief visual representation of inserting the human insulin gene into the plasmid of the E. coli cell [8].
Once the recombinant E. coli cells are identified and isolated transferred to large fermenters where they will be grown. Nutrients such as nitrogen, sugar, salt and water are in the broth to supply the E. coli cells for adequate growth. Ampicillin is also added to the broth to kill microbes that may have made their way into the fermentation tanks except for the desired ampicillin resistant E. coli. E. coli reproduces every 20-30 minutes this exponential growth continues until the E. coli reach a saturation point. The E. coli cells are allowed to reproduce for several days until they reach a certain concentration. The cells have been inhibited from producing insulin up to this point because of the repressor protein that has been sitting near the insulin gene. A chemical is then added to induce the production of insulin within the cells. It only takes a few hours for the cells to produce a high enough yield of insulin [9].
The cells are then harvested from the fermentation take and are centrifuged to separate the from the broth. The broth is separated from the cells and a chemical is added to break down the cell membrane and release the insulin from the cells. The insulin then must go through numerous purification steps before the 21 amino acid A-chains and 30 amino acid B-chains are mixed and joined together by disulfide bonds at a ratio of 1:1 [10] [11]. The insulin is then again purified before the final step which is crystallization. This is done by adding zinc and dehydrating the insulin to form it into a crystal structure before it is ready to be packaged and stored for distribution.

Figure 2. The Structure of Insulin. This shows the primary structure of the insulin that is formed after the 21 amino acid A-chain, and 30 amino acid B-chain are linked together by disulfide bonds [11].
Significance of Synthetic Insulin
The production of recombinant human insulin has saved the lives of many diabetics around the world. Insulin has become for available and affordable to people than before. It has allowed diabetics to live more normal live and continue throughout their days without having to worry too much about their blood sugar levels. The discovery of E. coli grown insulin has also paved the way for other recombinant hormones that are necessary for people with other diseases. For example, E. coli and other bacteria are also used to produce hormones for immune system repair, fertility, and blood production [12].
Conclusion
Whoever thought that e. coli would provide so many benefits such as the production of recombinant human insulin. This review has gone over how the human insulin gene is placed into the plasmid of the E. coli cell and how the insulin is then extracted and processed. It has also gone over why E. coli is the preferred organism for recombinant insulin production and its impact that it had on other recombinant hormones that are now produced.
Works cited

[1] “CDC Newsroom.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 18 July 2017, www.cdc.gov/media/releases/2017/p0718- diabetes-report.html.
[2] McClatchey , Forester. “A Brief History of Insulin.” Beyond Type 1, 31 Oct. 2017, beyondtype1.org/brief-history-insulin/.
[3] Baeshen, Nabih A, et al. “Cell Factories for Insulin Production.” Microbial Cell Factories, vol. 13, no. 1, 2014, doi:10.1186/s12934-014-0141-0.
[4] Pickup, John. “Human Insulin.” British Medical Journal (Clinical Research Edition), vol. 292, no. 6514, 1986, pp. 155–157. JSTOR, JSTOR, www.jstor.org/stable/29521896.
[5] “Playing catch-up with Escherichia coli: using yeast to increase success rates in recombinant protein production experiments” Frontiers in microbiology vol. 5 85. 5 Mar. 2014, doi:10.3389/fmicb.2014.00085
[6] Shin CS, Hong MS, Bae CS, Lee J. Enhanced production of human mini-proinsulin in fed-batch cultures at high cell density of Escherichia coli BL21(DE3) [pET-3aT2M2] Biotechnol Prog. 1997; 13:249–257. doi: 10.1021/bp970018m.
[7] Gurramkonda C, Polez S, Skoko N, Adnan A, Gabel T, Chugh D, Swaminathan S, Khanna N, Tisminetzky S, Rinas U. Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin. Microbe Cell Fact. 2010; 9:31. doi: 10.1186/1475-2859-9-31.
[8] Petrides, Demetri, et al. “Computer-Aided Process Analysis and Economic Evaluation for Biosynthetic Human Insulin Production—A Case Study.” Biotechnology and Bioengineering, vol. 48, no. 5, 1995, pp. 529–541., doi:10.1002/bit.260480516.
[9] “E Coli Containing the Insulin Gene Grow in a Fermenter.” Treating Diabetes, tacomed.com/chapter-12-recombinant-dna-production-of-insulin/e-coli-containing-the-insulin-gene-grow-in-a-fermenter/.
[10] Goeddel, David V., et al. “Expression in Escherichia Coli of Chemically Synthesized Genes for Human Insulin.” Proceedings of the National Academy of Sciences of the United States of America, vol. 76, no. 1, 1979, pp. 106–110. JSTOR, JSTOR, www.jstor.org/stable/69441.
[11] Gebel, Erika. “Http://Www.avensonline.org/Fulltextarticles/JSUR-2332-4139-S1-0001.Html.” Journal of Surgery, July 2015, pp. 01–07., doi:10.13188/2332-4139.s100001.
[12] Hua, Qing-Xin, et al. “Mechanism of Insulin Chain Combination.” Journal of Biological Chemistry, vol. 277, no. 45, 2002, pp. 43443–43453., doi:10.1074/jbc.m206107200.

Principles and Applications of Polymerase Chain Reaction

Principles and Applications of Polymerase Chain Reaction
Polymerase chain reaction (PCR) was created by Mullis in 1983 and ensured in 1985. Its standard relies upon the use of DNA polymerase which is an in vitro replication of unequivocal DNA plans. This technique can make a huge number of copies of a DNA piece from a DNA expel. If the plan of interest is accessible in the DNA to remove, it is possible to explicitly mirror it in incredibly immense numbers. The force of PCR relies upon the way that the proportion of cross section DNA isn’t, on a basic level, a compelling component. We can thusly improve nucleotide groupings from moment proportions of DNA remove. PCR is as such a strategy for purification or cloning. DNA removed from a living being or test containing DNAs of various sources isn’t analyzable. It contains numerous masses of nucleotide game plans. It is likewise critical to keep and sterilize the progression or groupings that are of interest, paying little mind to whether it is the course of action of value or noncoding game plan introns, transposons, littler than ordinary or microsatellites. From such a mass of groupings, that involves the system DNA, the PCR can along these lines pick in any event one progression and increase them by replication to a few billions of copies.
At the point when the reaction is done, the proportion of cross section DNA that isn’t in the domain of interest won’t have vacillated. Then again, the proportion of the strengthened sequence(s) (the DNA of interest) will be tremendous. PCR makes it possible to improve a sign from an establishment uproar, so it is a nuclear cloning strategy, and clone comes back to faultlessness. There are various uses of PCR. It is a system now essential in cell and sub-nuclear science. It awards, especially in several hours, the “acellular cloning” of a DNA piece through an automated system, which by and large takes a couple of days with standard methodology of sub-nuclear cloning. Of course, PCR is commonly used for characteristic purposes to recognize the closeness of a DNA progression of either life structure in a natural fluid. It is moreover used to make genetic fingerprints, paying little respect to whether it is the inherited distinctive verification of a person with respect to a lawful solicitation, or the conspicuous evidence of animal arrangements, plant, or microbial for sustenance quality testing, diagnostics, or varietal assurance. PCR is yet central for performing sequencing or site-composed mutagenesis. Finally, there are varieties of PCR, for instance, steady PCR, centered PCR, PCR in situ, RT-PCR, etc. The examination of regular flightiness is another wild that requires high throughput sub-nuclear development, quick and PC memory, better approaches to manage data assessment, and the compromise of interdisciplinary capacities.
PCR makes it possible to get, by in vitro replication, different copies of a DNA part from a concentrate. Cross section DNA can be genomic DNA similarly as equal DNA got by RT-PCR from a separation RNA remove, or even mitochondrial DNA. It is a system for getting a great deal of a DNA game plan from a DNA test. This escalation relies upon the replication of a twofold stranded DNA group. It is isolated into three phases: a denaturation arrange, a hybridization organize with foundations, and a prolongation arrange. The aftereffects of each blend step fill in as a design for the going with propels, along these lines exponential strengthening is cultivated. “The polymerase chain reaction is done in a reaction mix which contains the DNA evacuate, Taq polymerase, the fundamentals, and the four deoxyribonucleoside triphosphates (dNTPs) in bounty in a support game plan” (Kadri, 2019). The mechanical get together allows the programming of the term and the movement of the cycles of temperature steps. Each cycle consolidates multiple times of a few a few seconds. The methodology of the PCR is subdivided into three stages as seeks after.
The denaturation is the segment of the two strands of DNA, obtained by raising the temperature. The essential time period is finished at a temperature of 94°C, called the denaturation temperature. At this temperature, the cross-section DNA, which fills in as a structure during the replication, is denatured: the hydrogen bonds can’t be kept up at a temperature higher than 80°C and the twofold stranded DNA is denatured into single-stranded DNA. The consequent development is hybridization. It is finished at a temperature generally some place in the scope of 40 and 70°C, called starter hybridization temperature. Reducing the temperature allows the hydrogen bonds to change and, thusly, the correlative strands to hybridize. The presentations, short single-strand progressions correlative to regions that flank the DNA to be upgraded, hybridize more adequately than long strand system DNA. The higher the hybridization temperature, the more specific the hybridization, the more express it is. The expansion is the third time period is done at a temperature of 72°C, called extending temperature. It is the amalgamation of the comparing strand. At 72°C, Taq polymerase binds to arranged single-stranded DNAs and catalyzes replication using the deoxyribonucleoside triphosphates present in the reaction mix. The territories of the organization DNA downstream of the fundamentals are thusly explicitly joined. In the accompanying cycle, the pieces mixed in the past cycle are in this manner network and after several cycles, the pervasive species contrast with the DNA gathering between the locale where the preparations hybridize.
This is one of the most astonishing employments of PCR. It makes it possible to isolate, as it were, to channel a quality without relying upon standard techniques for nuclear cloning which involve in embeddings a DNA library in a plasmid vector which is then used to change a bacterial strain whose clones after decision are screened. The affirmation is much faster and significantly less discretionary using PCR. Acellular cloning is used when using PCR on the grounds that it is useless to use a cell structure (minute living beings, yeast, and animal or plant cell) to strengthen the clone. The affirmation of sub-nuclear cloning by PCR depends upon two huge criteria: the choice of DNA discrete (arrange DNA) and foundations. It is without a doubt essential to have basically strong data on the course of action of the quality that will be cloned and furthermore flanking progressions to fuse the plans of foundations principal for its upgrade in whole or somewhat.
On the other hand, is it still essential to play out the PCR on the fitting cross section DNA? We can pick the genomic DNA that fuses the hard and fast game plan of the genome and thusly all the characteristics of the species. For this circumstance, the characteristics consolidate the two exons and introns and their upgrade achieves the cloning of the all-out quality progression and in any occasion, dependent upon the preparations that have been picked, regulatory territories. Regardless, we can in like manner remove the diplomat RNA (mRNA), as it were, the fundamental coding progressions of the quality the transcripts. Since RNAs are inconsistent, conveyance individual RNAs are changed into comparing DNA (cDNA) by RT-PCR, a variety of PCR that usages reverse transcriptase and licenses changing the RNA courses of action into DNA.
It is on this cDNA library that PCR is then performed to clone the nature of interest. For this circumstance, the course of action is progressively stunning. The closeness of the quality transcript in the concentrate depends upon the cell type, tissue, or organ from which the mRNA extraction was performed. Without a doubt, translation is express to the cell type. Progressively veritable, the assertion of value is often coordinated by physiological segments, normal, for this circumstance the nature of interest isn’t generally translated, and the cDNA library may not contain it. Finally, it must be said that interpretation is itself controlled and is much of the time joined by elective uniting. This marvel prompts exon transfer at the hour of extraction of the introns and prompts the surge of different proteins from a comparative quality. It seeks after that depending upon the cell type and authoritative profiles, we may not be dealing with a comparative transcript. It is before long very interesting to clone a transcript since its nucleotide gathering identifies with the amino destructive course of action coming about due to the understanding.
The enlargement of genotyping approaches to manage each and every living structure has made imperative advances in the multiplication of the authentic setting of life. At the masses level, the movement and repeat of known inherited polymorphisms in a creature classification can highlight the propelling forces at play, reveal the effects of ordinary assurance, and accumulate measurement change. What’s more, the connection of the groupings of comparative characteristics between different species and that of whole genomes is at the origin of the nuclear phylogenies that correct presently win in the course of action. They make it possible to pursue the associations between species dependent on the difference of their DNA groupings. In that limit, the PCR is a key stage at two levels. The primary concerns the detachment of homologous characteristics in a couple of creature classifications and their depiction. The second is the age of improved full-scale genomic DNA for genome sequencing and comparative assessment.
References Kadri, K. (2019). Polymerase Chain Reaction: Principles and Applications. Online First, 10-50.

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