Wynne

Hi, my name is Wynne. I am going to be a junior this year at the Field School, in Washington, D.C.. In middle school, I actually used to really dislike science. It was the subject I dreaded most. However, I took a biology course in ninth grade that changed the way I look at science, specifically biology. I'm especially interested by the genetics and anatomy. I wanted to come to this camp to dig deeper into genetics and see if it is something I want to potentially begin a career in, as well as to have more experience and knowledge going into the coming school year. I'm excited to learn about synthetic biology, which is something I've never thought about! This year I took chemistry, and next year I plan on taking an advanced biology course. Outside of biology, I have an older brother, who will be a senior next year. I also have three cats and one dog (but I'm more of a cat person). I go to a small private school where there are about 70 people in each grade, which means I know everyone in my school a little too well. My favorite books are the Harry Potter series, and my favorite movie is Ferris Bueller's Day Off. I love soccer, and I play for my school, as well as out of school. I also play basketball and run track for my school. I have a passion for film photography and am taking classes in and out of school to become a better photographer. I also lead the community service club at my school and am part of the honor council, which deals with academic honesty. I look forward to having an awesome experience at BLI!


 * Pichia pastoris in Biopharmaceuticals **

Despite the successful uses of //E. coli// in the growing field of biopharmaceuticals, there is a need for simple cells that are able to be engineered to be more compatible with human cells. //E.coli//, while capable of producing many important therapeutic glycoproteins, is not capable of producing post-translational modifications. Scientists have identified //Pich////ia pastoris//, a methylotrophic yeast as being able to provide new capabilities to the field of biopharmaceuticals. The field of biopharmaceuticals is emerging in the world of modern medicine as a result of the increase in knowledge of biological engineering and synthetic biology. Biopharmaceuticals are recombinant therapeutic cells that have been created using biotechnology. These drugs are engineered to be more effective and to do things that drugs produced using only pre-existing biological material would not be able to accomplish. A well-known and successful example of the potential of biopharmaceuticals is recombinant insulin, human insulin made by using proteins secreted by //E. coli// instead of being made from animals. //E. coli// is an extremely useful bacteria in the field of biopharmaceuticals, as it is single-celled, rapidly reproduces, can be stored frozen and is relatively inexpensive to use. However, for all its benefits, //E. coli// has its limitations too. It is a prokaryote and is not able to perform post-translational modifications (PTMs) that are found in eukaryotes, such as glycosylation. PTMs are necessary for many fully-functioning, recombinant human proteins, as they determine how a protein is folded or cut, making a protein mature. There is a need for simple cells that are able to work as efficiently as //E. c////oli// and also perform necessary PTMs that make many proteins fully-functional in humans.

//Pichia pastoris// is a methylotrophic yeast that is widely used in biological engineering because it is eukaryotic and has strong, inducible promoters. Its two alcohol oxidase genes that have inducible promoters, AOX1 and AOX2, allow Pichia pastoris to use methanol as an energy source. These promoters are repressed by an excess of glucose. In synthetic biology, the desired genes can be inserted into the DNA and can be easily induced by the presence of methanol. It is able to grow in similar conditions to //Saccharomyces cerevisiae//, the most commonly used yeast cell in biopharmaceuticals, therefore there is no need for specialist equipment, and it is able to produce PTMs. The benefit to using //P. pastoris// over //S. cerevisiae// is that the PTMs in //P. pastoris// are much more similar to those in humans.

Additionally, //P. pastoris// does not secrete a large amount of endogenous protein, protein produced from the expression host itself. Therefore, there is less need to isolate foreign proteins, during the purification process. //P. pastoris// reproduces rapidly and can grow to a very high cell density because of its preference for respiratory growth, almost to the consistency of a paste, reaching more than 150 g dry cell wall per liter (see Figure 1). In 1993, the Phillips Petroleum Company in Bartlesville, Oklahoma released the //P. pastoris// expression system to academic science labs, leading to the extreme growth of knowledge that scientists now hold regarding the methylotrophic yeast. All of these factors make //P. pastoris// a good candidate for an expression system in the production of proteins in biological therapeutic medicine. It is more suited for the production of proteins in biopharmaceuticals than //E. Coli// or other known existing organism because it is a eukaryote, allowing it to be able to perform PTMs, specifically disulfide bonds and glycosylation, most similarly to humans.

 Figure 1. Growth curve of //Pichia pastoris// cells during the fermentation with NH4Cl/glucose–glycerol–methanol as nitrogen/carbon sources

The potential of //P. pastoris// with the technology of synthetic biology is extensive, especially in the three topics of glycoengineering, synthetic promoters, and molecular toolboxes (see Figure 2). Glycoengineering deals with glycosylation, the most complicated and the most common PTM. Glycosylation is the enzymatic process in which glycans are attached to proteins, creating glycoproteins. The term “glycan” refers to sugars or any assembly of sugars, either in free form or attached to another molecule. Very similar to carbohydrates and saccharides, glycans are essential in many therapeutical pharmaceuticals, often determining the efficiency and important determinants of the biological activity of a protein. Glycoproteins can be produced by animal cell walls, however, the amount that can be produced is very limited.

 Figure 2. Current synthetic biology approaches to //Pichia pastoris// in biopharmaceuticals.

This lead scientists to experiment with glycosylation in yeast cells to produce a larger yield of glycoproteins. However, glycans in humans and plants are very different, therefore there are risks with attempting to use human glycosylation in bacterial or yeast cells. The O-linked and N-linked glycosylation that occurs in P. pastoris differs from glycosylation that occurs in mammals. The addition of N-linked high mannose oligosaccharides to glycoproteins post-glycosylation done by yeast raises a risk because it could trigger immunogenic responses in mammals, causing the antigens in the blood to be quickly filtered out by the liver. In addition to problems that arise with PTMs, //Pichia pastoris// itself is not exactly the ideal host for therapeutic proteins. Though it is able to produce glycoproteins similar to those in mammals, it does not have the reliability that //E. coli// has. It must be prepared almost immediately before use, takes multiple days to work, and expression yields vary between clones. Often many clones must be tested for protein expression to find a satisfactory producer.

Because of these inconveniences, there has been research to try and eliminate hyperglycosylation in yeast cells, and add glycosidases and glycosyltransferases, transporters for sugars, and add biopathways for the sugars, essentially humanizing the glycosylation process. Additionally, it is important to be correctly positioned along the ER and Golgi apparatus, as the enzymes involved produce the substrates for the next one to be activated. This process has been accomplished in //P. pastoris// and has broadened horizons in the capabilities of this useful yeast. In order for the success of humanized-glycosylation in //P. pastoris//, synthetic glycobiology approaches were taken. Synthetically created glycosyltransferases and glycosidases were produced with “localization characteristics” that had the desired catalytic properties. By separating the N-terminal cytoplasmic tail, the stem region, and C-terminal catalytic domain of these type II membrane proteins, and fusing the different parts together, the scientists were able to create new combinations and variants of glycoysltransferases and glycosidases to be inserted into //P. pastoris// that are better suited for humans (see Figure 3). Over 600 variants were tested to determine which would function best to produce artificial glycosylation pathways in //P. pastoris//. These heterologous pathways enable scientists to be able to control the complicated process of glycosylation.



Figure 3. The different variants of glycosyltransferases and glycosidases and design strategies. On the left shows glycoysltransferases and glycosidases in their natural forms. In the middle shows the creation of the catalytic domains and CTSs, and the right shows the final possible combinations.

There have been several breakthroughs for humanizing glycosylation in //P. pastoris// made by scientists in the past several years. In 2006, nine synthetic genes were introduced to //P. pastoris//, and six native genes were deleted. This implemented the production of complex terminally sialylated glycoproteins. Because we are now able to control glycosylation in //P. pastoris// to a certain extent, glycoforms like one recently produced that has improved antibody-mediated effector functions can be created, paving the way for more advancements in the potential of glycoproteins. On a larger scale, there have been experiments where //P. pastoris// has acted as an expression system for an enzyme that could potentially target cancer cells. The possibilities for what will be able to be accomplished with //Pichia// //pastoris// are infinite, especially one the glycosylation process is completely humanized.

Work Cited

http://miller-lab.net/MillerLab/wp-content/uploads/pichia_system.pdf

http://www.pichia.com/wordpress/wp-content/uploads/2012/09/Christian-Julien-Bioprocess-2006-Pichia.pdf

http://www.pichia.com/wordpress/wp-content/uploads/2012/09/Christian-Julien-Bioprocess-2006-Pichia.pdf

http://www.piercenet.com/browse.cfm?fldID=4E12331D-5056-8A76-4E72-1C5A427505F1

http://wwwchem.csustan.edu/chem44x0/SJBR/amarjit.htm

http://www.sciencedaily.com/articles/b/biopharmaceutical.htm

http://www.sciencedirect.com/science/article/pii/S0958166913000384