Electronic Theses and Dissertations

Identifier

6202

Date

2018

Document Type

Thesis

Degree Name

Master of Science

Major

Chemistry

Concentration

Analytical Chemistry

Committee Member

Abby Parrill-Baker

Abstract

Nipsnap1 (4-nitrophenylphosphatase domain and non-neuronal SNAP25-like homolog 1) is a novel mitochondrial protein shown to interact with the intracellular domain of amyloid precursor protein (APP), one of the key biomarkers implicated in Alzheimer’s disease (AD) pathology. A formerly developed expression strategy for Nipsnap1 based on the pET-28a expression system showed direct binding of purified recombinant Nipsnap1 to NAD+ and NADP+. This was further confirmed using Circular Dichroism (CD) spectroscopy, indicating that NAD+ binding produces a structural change in Nipsnap1.To address the low yield of Nipsnap1 protein in the former expression system, several approaches and conditions were evaluated in this study. A GST-fusion strategy was developed to stabilize the recombinant protein. GST fusion did not result in sufficient solubility of Nipsnap1, with the majority of expressed GST-fused Nipsnap1 aggregating in inclusion bodies. Growth curve analysis showed that Nipsnap1 expression did not cause a bacteriostatic effect. After several experiments to optimize induction, solubilization and purification of recombinant Nipsnap1, the yield for Nipsnap1 remained low, but several truncated forms of Nipsnap1 were expressed in higher quantities. Future strategies of recombinant Nipsnap1 expression and purification may benefit from the use of a codon-optimized form of Nipsnap1 introduced into expression systems known to aid in protein solubility and stability.

Comments

Data is provided by the student.

Library Comment

Dissertation or thesis originally submitted to the local University of Memphis Electronic Theses & dissertation (ETD) Repository.

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