1. Investigating conformation specific drug targets of human Thymidilate synthase
Ukegbu B. Iroha
Department of Pharmaceutical sciences,
University of South Carolina, Columbia.
Mentor: Dr. Sondra Berger
December 10, 2004.

2. Introduction/Objective
Cancer
Thymidilate Synthase
Background
Aim of research

3. Thymidylate Synthase (TS) Reaction Pathway

4. Role of Thymidylate Synthase

5. Thymidylate Synthase Structure
Cys
180°
Cys
Nucleophilic Cys is located in the active site cleft

6. Native
Complexed

7. Constructing Mutant TS proteins
Method
Constructing Mutant TS proteins
JM109
Dpn I
digest
PCR
rxn
TX61-
catalytic
activity
Growth Conditions
+/- exogenous
Thymidine
Protein purification
FPLC
blue sepharose
sepharose Q
3-D structure determination
Ligand
Binding
analysis

8. Creating V3A mutant dH2O -37.5ml 10x Pfu Buffer -5.0ml DtNps -1.0ml
DPNI digest
PCR
dH2O ml
10x Pfu Buffer ml
DtNps ml
Templates ml
Primer1(SB229) -2.5ml
Primer 2(SB230)-2.5ml
Pfu Turbo ml
50ml
Mutation primer sequence position
V3A SB `-TTA TGC CTG CTG CTG GTT C-3`
SB `-GAA CAA GCA GCA GGC ATA A-3`

9. Transformation on [Tx61-] cells

10. Plasmid purification Grew 10ml overnight culture
Use Qiagen plasmid mini-prep purification kit protocol

11. Western blot analysis of Active and inactive stabilized mutants of hts in TX61- cells.
10ml overnight culture were sonicated in Buffer A
Remove cellular debris by centrifugation
Use bradford assay to determine concentration of total soluble protein
Load protein in well.
y = x
R2 =
Mutant protein SLB load 23 26 32 8 9 11 15 17 21
Hts
Tx61-
V3A

12. Future Direction Characterization of V3A Activity assay
Ligand binding (DUMP ligands)
Reactivity of Catalytic cystine
Structural Determination
Crystallization of protein.

13. Acknowledgement Dr. Sondra Berger Saphronia Johnson Brittnaei Bell
Dr. Omar Bagasra
Twaina Harris
Claflin University
University of South Carolina, Columbia
National Cancer Institute