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West UHC, BRR, seminar Presentation, Nick West.pdf

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https://ir.library.oregonstate.edu/concern/undergraduate_thesis_or_projects/44558g02f

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  • Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease where paralysis of the body is the result of motor neuron function loss. A potential cause of ALS is the loss of zinc from Superoxide Dismutase (SOD). SOD’s cellular responsibility is to enzymatically react with the superoxide anion forming oxygen and the less hazardous chemical species of hydrogen peroxide. In order for SOD to be stable and fully functional it must be bound to both copper and zinc. When SOD undergoes a variety of mutations, the specifically folded protein is more susceptible to lose the zinc atom. Research has shown the loss of zinc from SOD can activate SOD to create toxic intermediates that can stimulate motor neuron cell programmed death and ALS onset. The source and exact processes by which SOD receives its zinc atom has yet to be fully described. A potential family of proteins responsible for SOD’s zinc are the group known as the Metallothionein’s. Metallothionein’s are a family of metalloenzymes critical to regulating cellular zinc concentration levels. The combination of low molecular weight and the high amount of cysteine residues makes this protein exceptionally difficult to analyze in vitro and in vivo. Using recombinant DNA experimental techniques the Metallothionein isoform 2 gene was successfully inserted into the pTYB11 plasmid using restriction endonucleases EcoRI and SapI. Attempted expression of Metallothionein using the expression strain, BL21(DE3) pLysS showed unsuccessful induction of Metallothionein expression. However, utilizing the Shuffle T7 expression system provided some evidence that metallothionein isoform 2 was expressed. Thus, our present results seem to hint at an easier form of expressing Metallothionein in bacterial cells, but inherent flaws in the efficiency of the protein’s expression still exist.
  • Keywords: Protein Expression, Metallothionein, Restriction Endonucleases, Amyotrophic Lateral Sclerosis
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