Purification, Activity and Crystallization Of The SET10 Protein Open Access

Faraz, Shahdabul (2014)

Permanent URL: https://etd.library.emory.edu/concern/etds/gh93gz932?locale=en


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

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