Purification and Characterization of C. elegans Malate Dehydrogenase with the Use of Deae Ion-Exchange, Affinity and Gel Exclusion Chromatography

Date of Award

2008

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Keywords

C. elegans Purification, Enzymes, Malate Dehydrogenase

Area of Concentration

Chemistry

Abstract

This thesis continued the work of previous thesis students Ira Do (2000), Danny Gonzalez (2001), and Sarah Bondi (2002). For this thesis, malate dehydrogenase of Caenorhabditis elegans (MDH-1) was expressed in Escherichia coli using the pBAD/His A vector (Invitrogen). MDH-1 was purified using a newly developed DEAE ion-exchange resin followed by a Ni-affinity column. Based on results of gel filtration chromatography, the quaternary structure of native MDH-1 appears to be a dimer. Storing MDH-1 with the coenzyme NADH did not contribute to thermostability of the enzyme as shown by kinetic assays of the enzyme. The final purified MDH-1 had fewer contaminating proteins than samples prepared in previous research projects, but was significantly less concentrated. There were also indications that MDH-1 has a higher thermostability when the enzyme is more highly purified, as shown by kinetic assays of MDH-1 samples stored in thermal denaturation conditions. However, MDH-1 appeared to lose activity after repeated, rapid changes in temperature.

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