Date of Award

2020

Document Type

Campus Access Dissertation

Degree Name

Doctor of Philosophy in Chemistry (PhD)

Administrative Home Department

Department of Chemistry

Advisor 1

Tarun K. Dam

Committee Member 1

Lanrong Bi

Committee Member 2

Qinghui Chen

Committee Member 3

Shiyue Fang

Abstract

The existence of every living system depends on the concerted interactions of macromolecules. The major macromolecules include proteins, lipids, carbohydrates and nucleic acids. Numerous other molecules work in collaboration with them to keep the living systems functional. By studying macromolecular interactions, we gain insights into the specific pathways necessary for cellular survival and how these pathways are altered under pathological conditions. In order to study those molecular interactions and the biological processes they drive, one must obtain these (macro)molecules in pure form. Therefore, purification and separation techniques remain at the forefront of biomolecular and biomedical research. This dissertation presents new as well as modified techniques and shows their utility in purifying disease-related proteins and new bioactive compounds with therapeutic potential.

The first section of this dissertation describes a new protein purification method, termed Capture and Release (CaRe). This method was developed and validated to alleviate some of the limitations imposed by currently practiced techniques. CaRe uses the multivalent interactions that occur between a protein and its multivalent ligand to rapidly and effectively purify proteins from cell lysates and other crude preparations. CaRe was used to successfully purify three glycan-binding proteins or lectins (Galectin-3, SBA and ConA) and two glycoproteins (Tarin and Ovalbumin). This method does not need elaborate infrastructure and extended operational time that is required by conventional purification methods. CaRe is efficient, favorable even for small sample volumes, and could be a valuable tool for proteomics, glycomics, and glycoproteomics.

The second section of this dissertation shows the application of modified conventional separation techniques in the discovery of new bioactive compounds from plant tissues. The new compound is named Hely411, which is a hemolysin with an apparent molecular mass of 2 kDa. It interacts with N-linked glycoproteins and membrane lipids. Hely411 shows the characteristics of a glycoside with a constituent lipid moiety. It disrupts membrane structures before completely rupturing the cells. Hely411 could dissolve blood clots within 5 minutes of incubation and successfully produced RBC ghost cells. Considering all the characteristics, Hely411 could be used as a new therapeutic agent and a reagent for biomedical research.

Available for download on Friday, April 30, 2021

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