KKEK3151: Biochemistry

Semester 1, 2013/2014
Homework:

Insulin Production

Group 1 members:
1. 2. 3. 4. 5. 6. Kong Yu Pin Lee Pei San Lim Shun Yue Tan Kai Xian Teh Qian Hua Toh Beng Leong KEK110010 KEK110013 KEK110017 KEK110055 KEK110057 KEK110060

Lecturer-in-charge: Dr. Adeline Chua

Insulin is a hormone produced in the pancreas by the islets of Langerhans that regulates the amount of glucose in the blood (Insulin, 2010). People who lacks the hormone insulin cannot control their blood sugar levels and will suffer from the potentially fatal disease, called diabetes mellitus. Before biotechnology and genetic engineering were practiced, diabetic patients relied on insulin that is extracted from animals, such as pigs or cows, which proved effective to combat their illness. However, in some cases, the patient showed immune reactions and the body was unable to utilise the foreign insulin (Bliss, 1984). Therefore, genetic engineering was introduced to allow genuine human insulin to be produced by a bacteria, called Escherichia coli, which was genetically modified by inserting the human gene for the synthesis of insulin hormone into the bacteria (Madigan et. al. 2010). In the production of insulin, the product is, of course, the human insulin. It is a polypeptide with tertiary structure made up of 51 amino acids arranged in two chains that are connected by disulphide bonds; chain A has 21 amino acids, and chain B has 30 amino acids (Harrison et. al., 2002).

Figure 1: Tertiary structure of the polypeptide insulin, containing A and B chains connected with disulphide bonds.
Source: Madigan et. al., 2010

In a typical preparation of a recombinant plasmid for E.coli in the production of insulin, the raw materials required are the human genes encoding chain A and chain B, the plasmid of E.coli, the enzymes for the insertion of the genes into plasmid, such as EcoRI and ligase and the microorganism E.coli (Ladisch & Kohlmann, 1992). The genetically modified E.coli can be cultured on Luria-Bertani medium, which contained yeast extract, tryptone, inoculum broth, sodium chloride and several other trace metals (Tabandeh et. al., 2004). There are two main methods to produce recombinant human insulin from genetically modified bacteria, namely two chain method and proinsulin method. Currently, the proinsulin method is preferred due to the single fermentation and isolation steps involved, as opposed to the two fermentation and isolation steps involved in the two chain method, which consists of the separate production of the A and B-chains of insulin (Brown et. al., 2007). In the "proinsulin method", E.coli bacteria undergoes aerobic fermentation in which both A and B chains are translated and linked together by an extra peptide chain, called Cpeptide, forming a fused protein or proinsulin molecule. The formation of insulin from proinsulin follows two main steps (Bai et. al., 2003): folding & formation of disulphide bridges within the proinsulin molecule, and proteolytic cleavage with subsequent release of the connecting C-peptide. Within a bioreactor with optimum condition (pH 7, temperature of 37C), E.coli bacteria will produce proinsulin in the form of inclusion bodies, which are recovered by the disruption of cell and then solubilized. Insulin chains are released by cleaving proinsulin using cyanogen bromide, CNBr in 70% formic acid (Brown et. al., 2007).

Consequently, ion exchange chromatography, gel filtration and reverse phase chromatography steps are utilized to purify the product (Prasad, 2013). The overall process flow of insulin production is summarised in a flow diagram (Figure 2) below and illustrated in detail in Figure 3 in the Appendix.
Fermentation and In-situ filtration

Media preparation

Gel filtration

Crystallisation and dissolution

Chemical conversion

Insulin

Reverse phase chromatography

Crystallisation and dissolution

Ion exchange chromatography

Figure 2: Flow diagram of production of insulin. There are several problems that arose in this method. The foreign proteins made in bacteria must be isolated first by disrupting the bacterial cells and then purified. If the desired protein is contaminated with traces of bacterial proteins, this may cause an unwanted immune response in the body of the recipient of the protein. Furthermore, traces of lipopolysaccharide from the outer membrane of gram-negative bacteria, such as E.coli, are toxic towards human (Madigan et. al., 2010). The production of insulin is one of the many application of microorganisms, especially bacteria, to produce desired products. Industrial microbiology involves commercial products being produced on a large scale by microorganisms, which contrasts with biotech industry, whereby the products are typically made in relatively small amounts but are high in value (Madigan et. al., 2010). With the ability to use bacteria to artificially create substances needed by human being, the biological products can be industrially produced. The field of bioprocessing has extended from medicine and pharmaceutical products to food, agriculture, water treatment and polymers production. In short, insulin can be artificially produced by the recombinant bacteria, E.coli, by gene splicing, in which the human gene for insulin production is inserted into the plasmid of the bacteria. This will enable the bacteria to produce insulin in a nutrient-rich medium via aerobic fermentation. The insulin produced must be purified before it can be used by diabetic patients to prevent contamination.

References: Bai, Q., Kong, Y., & Geng, X.D. (2002). Studies of Renaturation with Simultaneous Purification of Recombinant Human Proinsulin from E.coli with High Performance Hydrophobic Interaction Chromatography. Journal of Liquid Chromatography & Related Technologies, 26(5), 683695. Bliss, M. (1984). The Discovery of Insulin. Chicago: University of Chicago Press. Brown, P., Fincher, M., Wadge, G., & Watson, A. (2007). Insulin Production Process Design. School of Biological and Agricultural Engineering, Louisiana State University. Harrison, R.G., Todd, P.W., Rudge, S.R., & Petrides, D. (2002). Bioseparations Science and Engineering. Oxford: Oxford University Press. Insulin. (2010). Oxford Dictionary of English (3rd ed.). Oxford: Oxford University Press. Ladisch, M.R., & Kohlmann, K.L. (1992). Recombinant Human Insulin. Biotechnology Progress, 8, 469-478. Madigan, M.T., Martinko, J.M., Stahl, D., & Clark, D.P. (2010) Brock Biology of Microorganism (13th ed.). San Francisco: Benjamin Cummings. Prasad, N.K. (2013). Downstream Process Technology: A New Horizon in Biotechnology. New Delhi: PHI Learning Pvt. Ltd. Tabandeh, F., Yakhchali, B., Shojaosadati, S.A., Khodabandeh, M., & Sanati, M.H. (2004). Growth Kinetics and Human Growth Hormone Production of a Heat-Inducible Recombinant Escherichia coli during Batch Fermentation. Iranian Journal of Science & Technology, 28(1), 12-17.

Appendix Media preparation

Water Raw material Other nutrients Inoculation Sterilization

Fermentation and In-situ filtration

Air filter

Air filter

Liquid waste

Gel filtration

Liquid waste Vapour

Crystallisation and dissolution

Impurities

Chemical conversion

Liquid waste

Ion exchange chromatography

Liquid waste Vapour

Crystallisation and dissolution

Impurities

Reverse phase chromatography

Liquid waste

Final products

Insulin

Figure 3: Overall process flow of insulin production.

Source: Prasad, 2013