Purification, Activity and Crystallization Of The SET10 Protein 公开
Faraz, Shahdabul (2014)
Abstract
The protein SET10, found originally in Schizosaccharomyces pombe, is a
member of SET domain proteins known for its methylation of side chains of lysine
residues. Although researchers have uncovered the activity and structure of various SET
domain proteins, not much is known about SET10. We, therefore, began to work with
this protein, focusing three main goals: to successfully purify the protein, to characterize
its activity, and to uncover pertinent structural information. We first transformed a
plasmid containing our SET10 gene into Escherichia coli bacterial cells. We then
induced the expression of this protein within the cells. After cellular lysis via sonication,
we obtained an impure solution containing our protein. We used several methods of fast
protein liquid chromatography (FPLC) to obtain our final "pure" solution.
To characterize activity, we first performed a peptide-pull down assay that
showed that SET10 was able to bind to the H3 peptide regardless of the methylation
status of the K14. We also performed mass-spectrometry experiments, which showed that
there was an increase in H3 peptide methylation activity with an increase in reaction time
and temperature. The monomethylation peaks, however, were small, suggesting that
perhaps SET10 was a slow-acting enzyme and/ or that the H3 peptide was not the ideal
substrate. Lastly, we performed a radiometric assay that showed that individual free
peptides were not ideal substrates for SET10. Rather, there was increased methylation
activity when using whole histones or octamers as substrates. Moreover, a variety of
different substrates were methylated by SET10, suggesting substrate non-specificity.
We then performed a trypsin digestion experiment, which showed that the SET10
protein did not have a smaller, more stable domain. We then set up crystallization screens
and were able to obtain crystal "hits" in two distinct conditions. Our crystals diffracted to
a resolution of 7A, which was not high enough to obtain atomic level detail. Our data,
however, allowed us to identify that the protein space group was P23 and that the unit
cell was simple cubic. Moving forward, our goal is to obtain higher quality crystals that
provide higher resolution diffraction data.
Table of Contents
Table of Contents
Section Pages
1) Introduction 1-2
2) Special Objectives and Steps 2-3
3) Results: Purification 3-8
4) Results: Activity 8-14
5) Results: Crystallization 14-21
6) Discussion 22-26
7) Materials and Methods: Purification 26-29
8) Materials and Methods: Activity 29-32
9) Materials and Methods: Crystallization 32-33
10) References Cited 34-36
11) List of Figures 36
About this Honors Thesis
| School | |
|---|---|
| Department | |
| Degree | |
| Submission | |
| Language |
|
| Research Field | |
| 关键词 | |
| Committee Chair / Thesis Advisor | |
| Committee Members |
Primary PDF
| Thumbnail | Title | Date Uploaded | Actions |
|---|---|---|---|
|
|
Purification, Activity and Crystallization Of The SET10 Protein () | 2018-08-28 12:36:25 -0400 |
|
Supplemental Files
| Thumbnail | Title | Date Uploaded | Actions |
|---|