Expression, Purification, and Analysis of Caenorhabditis elegans Mitochondrial Malate Dehydrogenase

Date of Award

2012

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Keywords

Biochemistry, c. elegans, Malate Dehydrogenase, Enzyme, Metabolism, Mitochondria

Area of Concentration

Biochemistry

Abstract

To this day Caenorhabditis elegans remains one of the most important and best understood model organisms in modern biological research. This nematode's small size, rapid reproduction rate, simple form, and ease of culture have rendered it crucial to the investigation of eukaryotic development and genetics. Key aspects of the organism's biology, however, have not been studied in detail. Mitochondrial malate dehydrogenase plays a key role in the Krebs cycle and gluconeogenesis, but remains poorly understood in C. elegans. Mounting a rigorous in vitro investigation in this area will require a method of producing the enzyme in sufficient quantities. Here, the Rosetta (DE3) host was used in conjunction with the IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) purification system to express and isolate an untagged MDH-2. This investigation sought to confirm the hypothesis that implementing the Rosetta strain would resolve previous difficulties in expressing the enzyme in vitro. The effort succeeded, achieving a substantial protein yield with minimal contamination. Enzyme activity assays were then carried out to analyze the kinetics of the MDH-2 catalyzed reduction of oxaloacetate to malate. Optimal conditions at pH 6-8 and 100 mM KCl or NaCl were determined. From Michaelis-Menten curve plots, KM values of 15±1 ?M oxaloacetate and approximately 50 to 70 ?M NADH with an overall specific activity in the range of 50 to 70 ?mol*minute-1*mg-1 are reported. These results should facilitate further investigations of this enzyme, and this purification method could be used to overexpress other metabolic enzymes.

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