Date of Award

5-2017

Degree Type

Honors College Thesis

Department

Chemistry and Biochemistry

First Advisor

Vijay Rangachari, Ph.D.

Advisor Department

Chemistry and Biochemistry

Abstract

Alzheimer’s disease (AD) is a common neurodegenerative disorder that affects people older than 65 years 1,2,5. It is characterized by the presence of extracellular plaque deposits that are seen specifically in the brains of AD patients 4,5. These plaques are mainly comprised of amyloid-β (Aβ) peptide aggregates. Aβ plaque production and deposition is believed to drive AD pathogenesis. Studying these proteins is crucial to understanding aspects of AD in order to develop possible therapeutic treatments. Recombinant expression of Aβ can also provide a handle to introduce mutations in the protein to further study their structure-function relationships. However, synthetic Aβ monomer protein is expensive, which can become an obstacle when studying Aβ’s biophysical and biochemical aspects. Therefore, in order to make it cost-effective, a standardization protocol for the expression and purification of Aβ from Escherichia coli (E. coli) cells is needed. Although expression of Aβ has been particularly difficult in the past, this project followed a recent report from Walsh et al. to express Aβ42 using inducible pET-Sac-Aβ(M1-42) plasmids. The E. coli cells were grown in LB media and collected. The cells were then lysed and Aβ isolated from inclusion bodies through sonication, centrifugation, urea solubilization. The urea-solubilized inclusion bodies were subjected to filtration through anion exchange chromatography using DEAE-cellulose beads. The monomer protein was separated from aggregates through size-exclusion chromatography. The identity monomeric Aβ was confirmed through MALDI-ToF MS. The pure monomer Aβ was then lyophilized and stored in HFIP (hexafluoro-2-propanol) until later use.

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