Research and Scholarship Grant
Title: Preparation of Noxo1 and Noxo1 Expression Vectors
PI: Nicole Y. Davis
Department of Chemistry and Physics
This proposal aimed to create expression vectors for two forms of a protein: Noxo1 and
Noxo1. Noxo1 (NOX Organizer 1) is a protein that serves as an “organizer” in a multiprotein enzyme complex that is involved in a wide range of cellular functions. Aberrant
function of these enzyme complexes leads to an array of diseases, including vascular
disease and certain cancers. Noxo1’s role as an organizer is accomplished through both
Noxo1-protein interactions and Noxo1-lipid interactions. The interest in these two forms
of Noxo1 is that they have identical sequences with the exception of the gamma form
having a five amino acid insert in the lipid-binding domain. Even though they are
sequentially very similar, the two forms have differences in how active the enzyme
complex is, where the proteins are located in a cell, as well as what tissues they are
expressed in. My research aims to study and characterize the protein-lipid interactions of
Noxo1.
A bacterial expression vector has been made that contains full-length Noxo1 with an
amino-terminal hexahistidine (His6) tag as well as a glutathione-s-transferase (GST) tag.
The presence of these tags will be utilized in protein purification in addition to serving as
a method of Noxo1 detection in functional assays. The expression vector also contains a
protease site, which allows both the His6 and GST tags to be removed from the protein.
The expression vector is being sequenced externally to confirm that we have the correct
sequence of amino acids. Once the sequence is confirmed, expression tests and purification
will begin immediately. I do not anticipate any obstacles with Noxo1 expression and
purification due to previous experience with this protein. While expression and purification
are carried out on Noxo1, mutagenesis will also be done to: create Noxo1which
requires inserting a five amino acid sequence), protein truncations (to examine isolated
protein domains to compare to full-length protein) and single amino acid mutations (to
determine the amino acid-specific interactions between Noxo1 and lipid molecules). Funds
from this grant were also used to purchase materials and supplies for lipid binding assays,
which will begin as soon as my lab has purified proteins.
This grant proposal served to aid in starting up my research. As I transition into a tenuretrack position, I am now working with undergraduates conducting research. The work
funded by this grant and downstream research will be presented by undergraduate student
authors at the Student Scholars Symposium at ASU in spring of 2016 and/or the 68th
SERMACS (Southeastern Regional Meeting of the American Chemical Society)
Conference in Fall 2016. The long-term goal is to have preliminary data for lipid binding
of Noxo1 and Noxo1 to submit as part of an external grant (NSF RUI) to investigate
what features of these two proteins leads to the variance in their functions by Fall of 2017.