Purification and Characterization of C. elegans Mitochondrial Malate Dehydrogenase

Emma Cassidy

Date of Award

2010

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Keywords

C. elegans, Malate dehydrogenase, Proteins, Biochemistry

Area of Concentration

Biochemistry

Abstract

C. elegans Malate Dehydrogenase (MDH-1) was isolated and purified from E. coli cells using an Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT) system (NEB). C. elegans is a small nematode worm which has long been considered an ideal model organism for scientific research due to it�s heartiness, transparency and relative simplicity. Malate dehydrogenase (MDH), which catalyzes the reversible conversion of malate into oxaloacetate, is a protein common to almost all living cells. It has been studied quite extensively due to its important role in several metabolic pathways, most notably the citric acid cycle. MDH has been characterized in many organisms, including bacteria and mammalian models; however, there has been very little research done on the C. elegans mitochondrial isoform, MDH-1. Better understanding of the structure and function of this enzyme may offer insight into the enzyme�s activities in higher organisms, which are more difficult to study. The C. elegans mdh-1 gene was ligated into a the plasmid vector pTXB1, adjacent to an intein sequence and a chitin binding domain (CBD), and transformed into ER2566 E. coli cells. The plasmid and cell design allowed for IPTG-induced expression of the target gene. The expressed MDH-1 was fused to the intein- CBD tag at its C-terminus. The intein is a peptide which cleaves at its N-terminus upon exposure to DTT. Cell lysate from induced cultures were applied to a chitin bead column, which bound the MDH-1-intein-CBD. DTT was then applied to the column to cleave the intein tags, and the MDH-1 was eluted from the column. The MDH-1 samples were sub jected to enzyme assays. at room temperature. The Km for oxaloacetate was 26.6 ?M, and the specific activity was 4.6 Units/mg.

Rights

This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.

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