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].

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      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.