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As the scientific director for a start-up Biotech company I have been asked to develop a system for the production of a protein therapeutic to treat a human autoimmune condition. I have thus studied about the various types of human autoimmune diseases and I have decided to treat Diabetes mellitus type 1 disease. In my following report I will be outlining the approaches and technologies that I would be using to produce a commercially viable product.
Human Autoimmune Diseases:
The body is mainly protected against microorganisms and other foreign substances by the immune system. The immune system is very important for proper functioning of the body and even a small change in the immune response can leave a person susceptible to infection. But the immune system itself can also cause disease by inappropriately attacking the body's own organs, tissues and cells causing prolonged inflammation and subsequent tissue destruction. Thus Autoimmunity can thus be described as the failure of an organism to recognise its own parts and which in turn allows an immune response against its own cells and tissues. Any disease that results from such an inappropriate immune response is termed as autoimmune disease.
Autoimmune diseases can be broadly classified into 2 general types
Systemic autoimmune disease ( Those that damage many organisms)
Localized autoimmune disease ( Those where only a single tissue or organ is damaged)
There are about 80-100 autoimmune diseases identified so far and these diseases are said to be chronic and life-threatening. The National Institute of Health (NIH) estimates that up to 23.5 million Americans suffer from autoimmune diseases and the prevalence is rising. Autoimmune diseases is also one of the top 10 leading causes of death in female children and women in all age groups up to 64 years of age.
Diabetes (medically known as diabetes mellitus) is the autoimmune disorder in which the body has trouble regulating its blood glucose levels. There are 3 main types of diabetes:
Type1: Type 1 diabetes is also called as Insulin dependent or Juvenile diabetes. This type covers almost 10% of all the diabetes cases. This type of diabetes is usually diagnosed in children and people under the age of 30. In this type the body stops making insulin.
Type 2: This Type 2 diabetes is also called as Non-insulin dependent or adult onset diabetes. This covers almost 90% of all the diabetes cases. Most cases of this kind are diagnosed in those over the age of 45 years. In this type the body makes its own insulin but the insulin made is thus either not sufficient for the body or it is that the body has become resistant to its effects.
Gestational Diabetes: It is a type of diabetes that is developed for the first time in some pregnant women, usually late in their pregnancy. This form of diabetes usually begins in the second half of pregnancy and it usually goes away after the baby is born. There are no major complications for this type of diabetes but people having gestational diabetes are usually at a higher risk of developing Type 2 diabetes later in their life.
Diabetes mellitus type 1:
Diabetes mellitus type 1 is also known as type 1 diabetes. It is a disease in which the pancreas produces too little insulin and the insulin that is produced is not sufficient to meet the body's requirements. Insulin is mainly required for the normal functioning of the body. Insulin is a hormone that regulates the metabolism of glucose (sugar) in the body. People with type 1 diabetes must take insulin injections to live. Insulin is produced by groups of cells in the pancreas, called the islets of Langerhans. These cells mainly release small amounts of insulin into the body. Every time a person eats, the glucose levels in the blood rises. This increase in the level of glucose in the blood triggers the cells in the pancreas to release the required amount of insulin. Insulin allows the blood glucose to be transported from blood into cells. Without insulin the blood glucose builds up in the blood and the cells starve of their energy source. The net effect is persistent high levels of blood glucose, poor protein synthesis and other metabolic derangements.
Figure 1: Statistics of treatment with insulin, tablets and diet medication among adults with diagnosed diabetes
[ Source: National Health Interview Survey 2006-2008]
Recombinant production of Human Insulin:
The first source of insulin that was used for clinical trials came from bovine (cow) and porcine (pig). The insulin from these sources were purified, bottled and sold. When this was administered to human beings some people found that this foreign protein caused allergy or other types of reactions and this led to a steady decline in the production of animal derived insulin. Biosynthetic "human" insulin came as a breakthrough and insulin is now manufactured widely for clinical use using recombinant DNA technology. Eli Lilly was the first to market such insulin, Humulin in 1982. Humulin was first produced by inserting an actual human DNA into a host cell (E.Coli).
Figure 2: Recombinant production of Human insulin
[Source: Understanding the Immune system with information provided by the National Cancer Institute and the National Institute of Allergy and Infectious diseases.]
Structure of Human Insulin:
Insulin is a small and simple protein that is produced in the cells of the pancreas called the islets of Langerhans. Insulin consists of two polypeptide chains A and B with a total of 51 amino acids. Insulin is one of the smallest proteins in the body. The Chain A is composed of 21 amino acids and chain B is composed of 30 amino acids [Insulin 1992]. These two chains A and B are linked by two inter-chain and one intra-chain disulfide bridge. In the human pancreas, both the chains are joined by connecting peptide (C-peptide) and the insulin is produced as a single polypeptide chain, proinsulin. [Mackin 1998].
. Figure 3: Human Insulin structure
[Source: Robert, J.F. Biochemistry and Physiology.]
Development of a system for the production of Human Insulin:
Mature insulin in its native conformation has not been produced so far in prokaryotic host cells using recombinant techniques. The reason being, the correct disulfide bond formation occurs only at the proinsulin. Several strategies have been developed for the production of proinsulin in E.Coli and these are commercially being used. [Tang and Hu, 1993; Kang and Yoon, 1991; Sung et al., 1986]. However, the critical cost factors are the complex purification processes and the formation of correct disulfide bonds during folding.
In this study we investigate the periplasmic production of proinsulin in E.Coli as a C-terminal fusion to DsbA. The bacterial periplasm is the most favourable compartment for the production of proinsulin. DsbA is the most important catalyst of disulfide bond formation and it is the most important oxidase of free sulfhydryl groups in the periplasm.
The E.Coli strains that were used are XL1 blue (Stratagene) and this was used as the host strain. The expression strains are C600 and RB791 (both E.Coli Genetic Stock Centre, New Haven). The cloning vector was pDsbA3 (Jonda et al., 1999). The source of the proinsulin gene is the vector pRK5-PI (Mather and Ullrich, 1987).
The plasmid preparation was carried out according to Sambrook et al. (1989). The cloning vector (pDsbA3-PI) was constructed by inserting the human proinsulin gene into pDsbA3. Additionally a 18 nucleotide sequence was introduced as shown in the figure.
Figure 4: Map of the vector pDsbA3-PI. The restriction sites used for cloning are indicated. Italics represent the linker sequence between DsbA (residues 1-189) and proinsulin (residues 196-281).
[Source: Winter, J. et al. (2000). Journal of Biotechnology.84.]
Now the E.Coli having the pDsbA3-PI was grown in LB medium. Once the optical density of the cells reaches 1 the cells are treated by osmotic shock resulting in periplasmic fractions. [Kang and Yoon, 1994]. These soluble periplasmic fractions and the residual insoluble proteins were analysed by SDS-PAGE. This human insulin contains an additional arginine residue. This arginine residue is found at the C-terminus of the B-chain. Insulin is released from the fusion protein by incubating it with 100Âµg trypsin for 10 min. The released insulin was then analysed by sandwich-ELISA. The amount of proinsulin produced in E.Coli can be calculated with the help of a standard curve created with native insulin.
In order to achieve efficient production of the fusion protein in soluble form with a native proinsulin part the expression was performed in E.Coli C600 and RB791 strains. The cells grew much faster in E.Coli RB791 than C600 resulting in higher cell density.
The yield of native proinsulin obtained from DsbA-proinsulin was 1000-fold higher than the secretory expression yield described for isolated proinsulin so far [Chan et al, 1981]. This indicates that DsbA is a very effective fusion partner for obtaining high amounts of soluble and correctly disulfide bridged proinsulin. In conclusion the data clearly indicates that the yield of human native proinsulin produced in E.Coli can be significantly increased by fusion of the proinsulin gene to DsbA. The great advantage of the fusion protein is that the proinsulin part can be cleaved off from the DsbA part by the addition of trypsin. Trypsin is commonly used for the conversion of proinsulin into insulin. By this proteolytic conversion it can be clearly shown that the proinsulin part of the fusion protein was folded correctly.
There are specific aspects that present potential limitations to the feasibility of the project and these are
Human Insulin may not be well accepted by some people and they may prefer to use insulin produced from animals.
The cost for producing Human insulin is almost double when compared to others so the cost may be a potential limitation.
During the development of Human Insulin there might be some problems with the post translational modifications.
Instability of the plasmid is also another limitation.
There might be problems in the folding of the disulfide bridge.
Biological activity and immunogenicity of recombinant protein may differ from the natural protein.
Secretes proteins only in the periplasm
But the greatest advantage of this system is that it gives us high quantities of the protein. Proteolytic cleavage events are a characteristic modification of many therapeutic proteins. Typically specific proteolytic enzymes cleave off the pro-peptide, yielding the mature protein. Proteolysis thus can also serve as a mechanism for releasing biologically active protein from an inactive precursor form. Proteolytic processing of proinsulin yields mature insulin product.
Figure 6: Proteolysis of Proinsulin into insulin.
[Source: Rhodes CJ et al. Insulin biosynthesis, processing, and chemistry. In: Kahn CR, Weir GC, King GL, et al, eds. Joslin's Diabetes Mellitus. 14th ed. Philadelphia, Pa: Lea & Febiger; 2005:65-82.]
Thus the proteolytic processing of proinsulin yielded mature insulin and this insulin can be given to patients who are suffering with Diabetes mellitus type 1 autoimmune disease. The system thus developed is mainly based on the recombinant DNA technology and this system gives us increased amounts of human proinsulin in the periplasmic space of E.Coli by fusion to DsbA. As the scientific director for a start-up Biotech company I would thus develop the above system for the production of Human Insulin for treating Diabetes Mellitus Type 1 autoimmune disease.