Type of Document Master's Thesis Author Dhar, Pratik Author's Email Address firstname.lastname@example.org URN etd-07122007-103329 Title Structural Studies of Lipoxygenases: Cloning, Expression and Purification of Lipoxygenases from Ananbaena and Fusarium Degree Master of Science (M.S.) Department Biochemistry (Biological Sciences) Advisory Committee
Advisor Name Title Marcia E. Newcomer Committee Chair Anne Grove Committee Member Sue G. Bartlett Committee Member Yong-Hwan Lee Committee Member Keywords
- anabaena fusion protein
Date of Defense 2007-06-21 Availability unrestricted AbstractLipoxygenases (LOX) are non-heme, non-sulfur, iron containing enzymes that catalyze the dioxygenation of unsaturated fatty acids. LOX enzyme is found in higher plants, some multicellular animals and some bacteria. In plants, the substrates for lipoxygenase enzymes are 18 carbon unsaturated fatty acids (linoleic and linolenic acids) while in animals, the substrate for this family of enzymes is the 20 carbon unsaturated fatty acid (arachidonic acid).
The LOX enzyme from the cyanobacterium Anabaena sp. strain PCC7120, which produces the smallest known LOX, and the fungus Fusarium verticillioides, which produces a LOX with dual specificity and remarkably high activity, were overexpressed using Escherichia coli (E. coli) bacterial expression systems. Both enzymes were also purified using conventional protein purification techniques such as affinity and size exclusion chromatography. Assays performed using linoleic acid as the substrate confirmed the purified enzymes to be in their active states and size exclusion chromatography confirmed sufficient purity for crystallization. The fusion protein from Anabaena, containing both LOX and AOS (allene oxide synthase), which is the naturally occurring form of the protein, has also been expressed using bacterial expression system.
New constructs encoding Fusarium LOX were engineered and cloned in pET-28b. The new constructs were designed to express protein lacking the T7 tag at the N-terminus, in an attempt to overcome crystallization problems. Both the new constructs have been successfully utilized to express the proteins, and protein obtained from one of the constructs has been overexpressed and purified.
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