Lara Bessa

Date of Award

2021

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Area of Concentration

Biochemistry

Abstract

Isocitrate dehydrogenase (IDH) is an important enzyme in the citric acid cycle that is responsible for catalyzing the forward and reverse oxidative decarboxylation reaction of isocitrate to alpha-ketoglutarate. This reaction also reduces NADP+ to NADPH in the matrix of the mitochondria. Mutations in the mitochondrial isoform of IDH have been seen in acute myelogenous leukemia and many other types of cancers. Although it’s unclear if these mutations are the primary cause of cancer cell growth, further study can aid in finding the connection between these mutations and cancer. Past thesis students have conducted studies on the wild type and mutant versions of the cytosolic IDH isoform, IDH-1, from Caenorhabditis elegans. These theses focused on the optimal conditions and purification of the protein. The mitochondrial isoform, IDH-2, is thought to have the same characteristics as its isoform IDH-1. However, there has been no research done on IDH-2 in C. elegans. This thesis focuses on the characterization and purification of IDH-2 from C. elegans. In this study, C. elegans IDH-2 was overexpressed in E. coli. It was found that the enzyme was not soluble in the native purification steps. After completing a protein solubility test, the enzyme was purified through a denaturing process with 6 M guanidinium and 8 M urea. In an attempt to refold the protein on the chromatography column, the urea was diluted out of the wash buffer. Protein assays were done in a variety of reaction conditions. Unfortunately, the protein was inactive due to the possibility of it not properly refolding during the purification.

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