Team:ULaVerne Collab/Design
How is insulin naturally synthesized and used in our body?
Insulin is a protein that is synthesized and secreted by beta cells which are located within Islets of Langerhans in the Pancreas. Its synthesis starts by transcribing the insulin gene to mRNA and then translating it to an inactive form of insulin known as Preproinsulin. Preproinsulin is then translocated to the rough endoplasmic reticulum (RER) by the N- terminal signal peptide. Once Preproinsulin is in the RER, its N- terminal signal peptide is cleaved off converting it to another inactive form of insulin called proinsulin. Two disulfide bonds are formed between the A chain and B chain of proinsulin. Proinsulin enters the Golgi Apparatus and then it enters immature insulin granules which function is to cleave the C-peptide converting proinsulin to an active form of insulin. Secretory vesicles containing the C-peptide, insulin, and amylin which is another hormone that regulates the rate at which food digest, is sent to the extracellular space due to the calcium presence when there are high blood glucose levels.
Once insulin leaves the beta cells, it travels through the bloodstream to the liver, muscles and adipose tissues in order to aid into increasing the glucose levels inside the cells. When a monomer insulin form, it binds to the insulin receptor located on the outer membrane of skeletal muscle cells, fat cells and liver cells. When insulin binds to the alpha subunit IR, it triggers the autophosphorylation of the phosphotyrosine binding (PTB) of the beta subunit due to a conformation change in the IR. Once the insulin receptor substrate is activated, it starts a signal transduction pathway which activates the phosphoinositide 3-kinase (PI3K) and this activates different kinases such as protein kinase B. Protein kinase B (PKB) activation triggers the translocation of glucose transporter (GLUT4) which acts as a channel for glucose to enter the cell. PKB activation starts glucogenesis process, which purpose is to reduce the blood glucose concentration in the bloodstream and store glucose. Also, glycolysis takes place which ultimately breaks down glucose into pyruvate to be used as energy.
Figure 1. Diagram demonstrating mechanism of Insulin
Type I Diabetes & Type II Diabetes
Diabetes mellitus is a disease affecting millions of people worldwide due to genetics, or lack of living a healthy lifestyle. Type 1 diabetes is an autoimmune disease that destroys the beta cells in the pancreas. Therefore, insulin is unable to activate the tyrosine kinase signaling pathway in the liver, fat and muscle cells to allow glucose to enter the cells to be used for energy or storage. However, type 2 diabetes could develop due to obesity. Our bodies start to be insulin resistance which means that cells are unable to open their GLUT4 channel to allow glucose to enter the cell and to use glucose as energy. Therefore, beta cells are constantly producing insulin since there is a saturation of glucose in the bloodstream. However, the insulin that beta cells produce is not “high quality”. For that reason, insulin is unable to activate the insulin receptors that will eventually allow glucose to get in the cells. Since the beta cells are overworking, they get tired and eventually they will die, and no insulin will be produced in the body.
For this reason, patients with diabetes must inject insulin to themselves throughout the day in order to allow their liver cells, skeletal muscle cells and adipocytes to used glucose as a form of energy and control their sugar levels in the bloodstream. However, insulin skyrocketing price makes it difficult to acquire since there is a monopoly on the market of insulin which means that certain people control this market. Once new insulin is developed, scientists put a patent on the insulin which increases its price. Also, the purification process used by manufactures is expensive as well. Therefore, we decided to come up with this project to synthesized cheaper insulin that will not be easier to purified and that could be accessible to everyone.
Design Goal
Our goal for this project is to synthesize both long-lasting and fast-acting single-chain insulin analogs in BL21 E. coli cells. The modifications we chose to incorporate into our protein synthesis include isoelectric point changes to the A and B chains and various linker lengths. The following is a guideline to how we approached our project design.
Engineering Principles
When constructing our parts, the main applications that we chose to focus on were changing the isoelectric point, adding a linker, and protein conformation. Native insulin consists of two polypeptide chains called the A chain and the B chain. These chains are held together by a C-chain that pushes the A and B chains together to form disulfide bonds. The C-chain is later cleaved to produce active insulin that has an isoelectric point between 5.30-5.35.
Figure 2. This image shows the inactive form proinsulin converting to an active form of insulin by cleaving the C-peptide.
Human insulin has been synthetically produced in E. coli and yeast since the early 1980s. During this manufacturing process, the A and B chains of insulin are produced separately and are later joined together to form disulfide bonds, resulting in an active and pure insulin protein. In this process, you omit the step in which you would have to cleave the C-chain, which also eliminates the need for any cleavage enzymes.
In the 1990s companies began to genetically modify insulin to create long-lasting and fast-acting versions of insulin. To create these insulin analogs, scientists began changing amino acid sequences on insulin, which would cause the isoelectric point to increase or decrease, as well as slightly change the structure of insulin.
When constructing our parts, the main applications that we chose to focus on were changing the isoelectric point, adding a linker, and protein conformation. We decided to use linkers between 8-12 amino acids long for two reasons:
Experimental Plan
Figure 3. Work flow to characterize proinsulin and single chain insulins.
We expressed each of our insulins in BL21 Escherichia Coli using a periplasmic expression system since it is a lowe cost effective host that is known to produce recombinant proteins. However, there are some limitations to obtain our desire proteins. E.coli is known to be a gram negative bacteria which contains an more oxidative environmental located in the periplasmic membrane. This environmental aids in the formation of disulfide bonds which is one of the biggest challenges of our project. Therefore, the Ecotin tag purpose is to translocate our proinsulin and single chain insulins to the periplasmic membrane of BL21 E. Coli hoping that the disulfide bonds and the folding of our proteins are correct. Once our circuits are located in the periplasmic membrane, we need to perform a lysing protocol to burst the outer membrane of the cell. We used CelLytic B reagent by Sigma Aldrich to break the cells open. However, the consequences about this protocol is that other proteins from E.Coli would be added to our solution. However, our purification process is designed to isolate our target protein which is proinsulin and single chain insulin
Figure 4. Diagram illustrating cell lysis protocol
Purification Process
Our proinsulin and single chain insulin constructs are designed with a Histidine tag in between some GSS linkers that were added to increase flexibility to the circuit. Adding those linkers will decrease the steric hindrance between proteins. The nickel purification process is an easy and low-cost protocol. The cell lysate supernatant is added to the Ni-NTA beads and all other proteins not containing our circuit should be washed off and discarded. Adding an elution buffer will allow our proinsulin protein or single chain insulins to detach from the beads. Therefore, the supernatant can be collected and loaded into an SDS page gel for further analysis.
The elution supernatant collected from the Ni-NTA must be cleaved off at the TEV site to isolate our proinsulin and single chain insulins. For our control circuit containing proinsulin, we plan on cleaving the C- peptide off by using trypsin.
Figure 5. Diagram representing Nickel purification in order to isolate proinsulin and single chain insulins from other proteins coming from E.coli
Characterization
Elisa Assay
The first step to characterize our insulin protein and single chain insulins, we need to determine if the folding of our proteins is correct and find out what is the concentration of our protein as well. Therefore, we plan on performing a Sandwich Elisa Assay which consists on using antibodies that bind to proinsulin with high specificity. If the wells turn a different color it would mean that insulin is present in the sample. The brighter the fluorescence, the higher the amount of insulin the sample.
Glucose Uptake Assay
After testing the conformation and concentration of insulin, we plan to perform a Glucose Uptake Assay using Chinese Hamster Ovary cells (CHO cells). The purpose of this assay is to find out if our insulins are still able to attach to the receptor. In addition, we would want to quantify the amount of glucose entering the cell.
More information about our circuits in our Design Phase one & Design Phase Two pages.
References
Ye, H., Xie, M., Xue, S., Charpin-El Hamri, G., Yin, J., Zulewski, H., & Fussenegger, M. (2017, January). Self-adjusting synthetic gene circuit for correcting insulin resistance. Retrieved June 2019, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5412959/.
Vieira. (2017, April 19). Amylin: the Other Hormone You Don't Produce in Diabetes. Retrieved June 2019, from https://www.diabetesdaily.com/blog/2014/03/amylin-the-other-hormone-you-dont-produce-in-type-1-diabetes/.
Type 1 Diabetes and Insulin. (2019, February 26). Retrieved June 2019, from https://www.endocrineweb.com/conditions/type-1-diabetes/type-1-diabetes-insulin.
Hooper, C. (2019, September 26). Overview of insulin signaling pathways. Retrieved June 2019, from https://www.abcam.com/pathways/overview-of-insulin-signaling-pathways.
Gradel, A. K., J., H. H., Lykkesfeldt, undefined undefined, Kristensen, undefined undefined, & F., undefined undefined. (2018, July 4). Factors Affecting the Absorption of Subcutaneously Administered Insulin: Effect on Variability. Retrieved June 2019, from https://www.hindawi.com/journals/jdr/2018/1205121/.
Diagnostics, C. (2017). Resources. Retrieved June 2019, from https://www.creative-diagnostics.com/insulin-signaling-pathway.htm.