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The Making of Insulin in Humans and Humanized Yeast

Christian Walleck 10/17/13

(Courtesy anthanycolpo.com)

Audience and Scope

This document provides an overview of insulin production in human pancreatic cells and yeast cells. It starts by explaining each step of insulin formation in humans from gene to active insulin protein. It also explains how yeast cells were manipulated by biologists to produce the human version of insulin The intended audience is freshman biology, biochemistry, or biomedical students. It is written assuming the audience has a high school level understanding of biology; therefore some basic terms are not explained. New students are often unsure how their major relates to the business world. This document provides an example of a highly developed process showing students one way biology is applied.

Introduction: What is Insulin and how does it work?

Insulin is a small protein secreted by the pancreas that signals cells throughout the body to absorb sugar.

Figure 1: Six insulin molecules together in 3D structure (Courtesy wikipedia.org/wiki/Insulin)

Insulin works through a series of steps: 1. Insulin is released in to the blood stream by pancreatic cells when blood sugar rises. 2. It induces cells to take up glucose by activating glucose transporters. 3. Glucose transporters move glucose in to cells until blood sugar returns to normal. Glucose transporters are proteins that fit through the plasma membrane of a cell allowing glucose to pass through the membrane. They are turned on or off by insulin to control the amount of sugar inside and outside the cell.

Figure 2: Glucose transporter. When active, it pumps glucose through the plasma membrane via an internal channel. (Courtesy
telstar.ote.cmu.edu/biology)

3 What causes Diabetes and How is it Treated? Insulin deficiency, known as diabetes, is a condition where too little insulin is produced to control blood sugar levels, or the insulin is mutated and loses its function. In either case, a patient with diabetes may need insulin injections as treatment. Injectable insulin came from pigs before synthetic insulin was invented. It was harvested by squeezing the pancreas from slaughtered pigs, a process similar to squeezing an orange for orange juice. Pig insulin is closer to human insulin than any other type; it differs by a single amino acid.

Figure 3: Porcine insulin (left) is identical to human insulin (right) except for a single amino acid. The structures remain almost identical.
(Courtesy rcsb.org/pdb/101)

Today, insulin is formed synthetically in yeast cells. The process was first perfected in e coli but later transferred to yeast because of better production capabilities.

How a Pancreas Cell Makes Insulin

Insulin production is a multi-step cellular process that follows three basic steps: transcription, translation, and modification. Each step of insulin synthesis is explained below. 1. Transcription: Converting DNA to RNA Cells begin insulin production by moving an RNA polymerase enzyme to the insulin gene (INS). RNA polymerase is the enzyme responsible for creating an RNA copy of a gene. It moves along the gene, making RNA as it goes. Once the INS gene is copied, the complete INS RNA molecule is free to leave the cell nucleus.
Figure 4. An RNA polymerase molecule moving down DNA, copying a gene as RNA.
(Courtesy http://hyperphysics.phyastr.gsu.edu)

4 2. Translation: Converting RNA to Protein RNA is converted to protein by a ribosome. Ribosomes are large, structured globs of protein and ribosomal RNA (rRNA) that read RNAs and convert them to proteins. Ribosomes are like translators. They take the language of RNA, which the cell cannot use directly, and convert it to proteins used for most cellular functions. The INS RNA codes for a protein called preproinsulin, an inactive form of insulin.

Figure 5. Ribosome structure. RNA enters from the bottom, is translated, and protein leaves out the top.
(Courtesy rcsb.org/pdb/101)

3. Modification: Conversion From Inactive to Active Insulin Preproinsulin must be modified to become active insulin. It is much larger than insulin, containing a signal sequence and three other sequences labeled A, B, and C peptides. The A and B peptides bond to each other to add stability.
Figure 6. Structure of preproinsulin Green- Signal Sequence Red- B peptide Blue- A peptide Orange- C peptide

(Courtesy hindawi.com/crim/genetics)

The signal sequence allows preproinsulin to enter the endoplasmic reticulum (ER), a small membrane structure in the cell. It activates a trans-membrane protein that pushes preproinsulin in to the ER. As it enters, the signal sequence is removed from preproinsulin resulting in proinsulin. Proinsulin moves from the ER to the golgi apparatus, another organelle. The golgi is like the post office of the cell. It sends molecules to the correct destination using their signal

5 sequences like an address. Molecules that do not have a signal sequence, such as proinsulin, are automatically placed in miniature bubbles called vesicles to be secreted by the cell.

Figure 7. Proinsulin moves from the ER (red) to the golgi apparatus (blue). It is placed in a vesicle where it is cut into insulin and then released by the cell. (Courtesy design4evolution.net)

Once inside the vesicle, enzymes called peptidases cut the C peptide out of proinsulin. The result is active insulin with two peptide chains. Finally, the vesicle reaches the cell surface and insulin is secreted.

Bullying Cells: How to Force Yeast to Make Human Insulin

The challenge of synthetic insulin production is forcing a different cell species to produce insulin unnecessarily. Like many students, cells are lazy. They only make enough proteins to survive. Yeast cells have no use for human insulin so they need to be coaxed in to producing it. Phase 1: Making the Recombinant DNA Recombinant DNA technology became possible as genes were isolated and sequenced for the first time. Recombinant DNA refers to any DNA cut by scientists and pasted together again in a specific sequence. INS gene requires modification to function in yeast. Yeast lack the enzyme needed to remove the signal sequence from preproinsulin so the entire signal sequence is deleted from INS gene. The INS gene is modified and moved to a circular piece of DNA called a recombinant DNA plasmid. A plasmid is a miniscule circular piece of DNA making it easier to insert in to a cell (chromosomal DNA would be nearly impossible). The plasmid MUST contain an origin of replication, the site on DNA that allows for DNA replication. Without an origin of replication, the cell would divide without replicating the plasmid and new cells would lose the ability to make insulin.

6 INS gene modification steps are listed. 1. 2. 3. 4. The INS gene is isolated from thousands of other genes. The signal sequence in INS gene is cut out by enzymes called endonucleases. The modified INS gene is inserted in a DNA plasmid containing an origin of replication. The recombinant DNA plasmid is finally inserted in yeast.

Figure 8. The process of making recombinant DNA

(Courtesy accessexcellence.org)

Phase 2: Inserting DNA in to Yeast Yeast cells were chosen as the cell species for human insulin production because they are common, easy to grow, and contain some of the cell machinery required to process insulin. Transferring the recombinant insulin plasmid to yeast is difficult because cells protect against invasion well. They need to recognize and destroy foreign molecules to prevent illness. Therefore, a transformation reaction must be used to insert DNA successfully. A transformation reaction is when the plasma membrane is weakened allowing foreign DNA to enter a cell without being destroyed. This occurs in three steps, neutralization of the plasma membrane, cooling, and heat shocking. Each step is explained below. 1. Neutralizing the Plasma Membrane Plasma membranes are designed to keep unwanted molecules out of the cell. The membrane is a phospholipid bilayer, seen in figure 9. The membrane has a negative charge on the outside which would repel DNA, also negatively charged. Calcium ions (Ca2+) are added to the cells in solution to neutralize this negative membrane charge. The Ca2+ ions line the outside of the membrane reducing the negative charge. As a result, DNA can pass through the membrane easier once heat shocked.

Figure 9. Plasma membrane structure (Courtesy studyblue.com/notes)

2. Cooling The cell solution is cooled for an hour to thicken their membranes. Plasma membranes are made of fatty acids just like butter or oil. When cooled, they become stiff like cold butter prompting the cell to adjust. Cells react by sending shorter fatty acids to the plasma membrane in an attempt to make it fluid again. Shorter fatty acids are present in olive oil. Even when cooled, they remain much more fluid than longer fatty acids. 3. Heat Shocking Heat shocking is the increase in cell temperature from about 40F to 100F in less than 1 minute. The plasma membrane, made up of short fatty acids due to cold temperatures, becomes excessively fluid. The recombinant DNA plasmid floating in solution can enter the cell through holes formed in the plasma membrane because of this hyper fluidity. The cells are cooled back to room temperature so they dont burst. They now contain the recombinant DNA plasmid to produce human insulin. Conclusion Insulin synthesis is surprisingly complex for a small protein. It requires numerous synthesis and modification steps before it functions properly. A continuous error within even a single step of the process often leads to diabetes. Luckily, synthetic insulin production has greatly increased output of medical quality insulin over the last thirty years. Biological understanding and engineering have transformed squeezing porcine insulin from pig pancreas in to a streamlined production process utilizing yeast cells as tiny factories. Today, synthetic insulin is a multi-billion dollar industry worldwide.

Works Cited
Cold Spring Harbor Laboratory, . How Insulin is Made Using Yeast. N.d. Infographic. DNA Learning CenterWeb. 11 Oct 2013. <http://www.dnalc.org/view/15929-How-insulin-is-made-usingyeast.html>. Langreth, Robert. "The $15 Billion Biotech Blockbuster Whose Sales Are Going Up With No End In Sight."Forbes. 02 10 2011: n. page. Web. 10 Oct. 2013. <http://www.forbes.com/sites/robertlangreth/2011/02/10/the-15-billion-biotech-blockbusterwhose-sales-are-going-up-with-no-end-in-sight/>.