Category: Defenses

Michigan Technological University
Department of Chemistry
John Hausman
Advisor: Dr. Ashutosh Tiwari
Master’s Defense
Monday August 10, 2015
9:30 am, Chem-Sci 101

ON THE PROTECTIVE PROPERTIES OF GLYCINE BASED OSMOLYTES IN A THIOL REDUCING ENVIRONMENT

The protective properties of osmolytes have been studied intently for decades. Originally used to aid in the crystallization of proteins in x-ray diffraction studies, these cosolvents have been shown to reverse protein denaturation and aggregation. Osmolytes aid extremophiles in surviving harsh environments by preferentially excluding themselves from the surface of the protein, thus directing water molecules to the protein’s surface. Their use is increasing and affects the everyday consumer as these osmolytes are naturally found in many health foods and also in many daily use products such as shampoo. Consumers also supplement their diets with thiol-based antioxidants such as glutathione as part of healthier life style. However, thiol-based antioxidants in high concentration have been known to cause un-intended health issues that relate to its disulfide reducing property. The disulfide-reducing agent can cleave the proteins’ disulfide-bonds and promote misfolding and aggregation. In this thesis, we investigated the influence of commonly consumed glycine-based osmolytes on providing stability to proteins against a disulfide-reducing agent. We chose glycine, sarcosine (N-methyl glycine), di-methyl glycine (DMG), and betaine (N,N,N-trimethylglycine) with increasing number of methyl groups. An added benefit to studying this class of osmolytes is to also investigate the impact N-methyl substitution has on the osmolytes’ protective properties. We studied the effect of these osmolytes on protein aggregation using spectroscopic techniques such as UV-visible absorbance, intrinsic fluorescence and extrinsic fluorescence measurements. In addition, we carried out non-reducing SDS-PAGE to check for higher order aggregates and characterized morphology of these aggregates using scanning electron microscopy. Overall, our results show that of all the osmolytes used, glycine was the best stabilizer followed by sarcosine. Betaine and dimethylglycine did not provide effective protection against disulfide-reducing influence.

Durga Pokharel

Advisor: Dr. Shiyue Fang

Doctoral candidate, Department of Chemistry

PhD Defense

Friday December 12, 2014   9:30am     Fisher 130

NON-CHROMATOGRAPHIC PURIFICATION OF SYNTHETIC BIOOLIGOMERS

Abstract

Synthetic oligonucleotides and peptides have found wide applications in industry and academic research labs. There are ~60 peptide drugs on the market and over 500 under development. The global annual sale of peptide drugs in 2010 was estimated to be $13 billion. There are three oligonucleotide-based drugs on market; among them, the FDA newly approved Kynamro was predicted to have a $100 million annual sale. The annual sale of oligonucleotides to academic labs was estimated to be $700 million. Both bio-oligomers are mostly synthesized on automated synthesizers using solid phase synthesis technology, in which nucleoside or amino acid monomers are added sequentially until the desired full-length sequence is reached. The additions cannot be complete, which generates truncated undesired failure sequences. For almost all applications, these impurities must be removed. The most widely used method is HPLC. However, the method is slow, expensive, labor-intensive, not amendable for automation, difficult to scale up, and unsuitable for high throughput purification. It needs large capital investment, and consumes large volumes of harmful solvents. The purification costs are estimated to be more than 50% of total production costs. Other methods for bio-oligomer purification also have drawbacks, and are less favored than HPLC for most applications.

To overcome the problems of known biopolymer purification technologies, we have developed two non-chromatographic purification methods. They are (1) catching failure sequences by polymerization, and (2) catching full-length sequences by polymerization. In the first method, a polymerizable group is attached to the failure sequences of the bio-oligomers during automated synthesis; purification is achieved by simply polymerizing the failure sequences into an insoluble gel and extracting full-length sequences. In the second method, a polymerizable group is attached to the full-length sequences, which are then incorporated into a polymer; impurities are removed by washing, and pure product is cleaved from polymer. These methods do not need chromatography, and all drawbacks of HPLC no longer exist. Using them, purification is achieved by simple manipulations such as shaking and extraction. Therefore, they are suitable for large scale purification of oligonucleotide and peptide drugs, and also ideal for high throughput purification, which currently has a high demand for research projects involving total gene synthesis. The savings with the new techniques compared with HPLC are estimated to be 70% to 90% depending on purification scale and throughput. We expect these new oligonucleotide and peptide purification technologies to be widely used in academic research labs, biotechnology companies, and pharmaceutical companies in the near future.

Mr. Robert K Brown

Advisor: Dr. Tarun K Dam

Master’s Candidate Department of Chemistry

Michigan Technological University

“Purification and Carbohydrate Binding Properties of Two New Plant Proteins”

Friday, December 12, 2014

10:00 – 11:00 AM 

Room 404 ~ Administration Building
Purification and Carbohydrate Binding Properties of Two New Plant Proteins

Abstract:

Protein glycosylation is an important post-translational modification for many biological processes such as cell recognition, intercellular communication and cell death. Proteins that are able to bind to glycosylated proteins via carbohydrates are called lectins. Hemolytic lectins are proteins or glycoproteins that undergo specific interactions with cell surface carbohydrates and subsequently induce cellular lysis. They are termed “hemo” lytic because of their ability to lyse erythrocytes. We have isolated a novel hemolytic lectin named HelyX from the bulbs of a monocot plant, as well as a mannose-binding lectin named DIL from a separate monocot species. HelyX is a uniquely robust hemolytic lectin. It shows concentration dependent reversible hemolytic/agglutinating properties against both human and rabbit erythrocytes. The activity was found to be carbohydrate dependent. HelyX was isolated using ammonium sulfate precipitation, size exclusion chromatography, and analyzed by gel electrophoresis. DIL was purified using a modified version of a newly developed protocol. DIL interacts with the plant enzyme invertase with high affinity. This high affinity interaction suggests that the binding site of DIL is complimentary to glycoproteins containing larger high mannose glycans. Invertase is central to plant metabolism and defense. Therefore DIL might play a modulatory role in plant metabolism and defense through its interaction with invertase. HelyX and DIL did not show lytic activity on free living amoeba, Acanthamoebae. Instead the lectins promoted cyst formation of amoeabae trophozoites indicating a lectin-mediated rearrangement of membrane architecture. This result indicates that the lytic activity of HelyX or DIL depends on the macromolecular landscape of the cell membrane.