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Plasmodium falciparum thymidylate kinase
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Targeting Thymidylate Kinase in Plasmodium falciparum (PfTMK)Riley Xu and Farah Abdelaziz
Thymidylate Kinase Thymidylate kinase catalyzes the
phosphotransfer from ATP to TMP
40% sequence identity to human TMK
Plasmodium
Human
Project Create a drug that disables PfTMK
Use nuclear magnetic resonance to find compounds that bind
Create allosteric inhibitor
No ligand
Ligand bound
Nuclear Magnetic Resonance Tag valines, leucines, and isoleucines
Assign peaks using mutations
Tells us how ligand binds
Mutagenesis Generate pET plasmid with PfTMK gene
Mutate wild-type gene with PCR mutagenesis
Mutations chosen to minimize structural change
Isoleucine to valine
Valine to alanine and isoleucine
Leucine to isoleucine
QuickChange Site Directed Mutagenesis
Uses complementary primer pairs that contain desired mutation
Linear amplification
Newly strands can’t be used as templates due to nicks
QuickChange Site Directed Mutagenesis (Agilent)
Failure to amplify newly synthesized strands
PfuUltra II QuickChange Mutagenesis Limited success using 50% higher Mg2+ concentration (2mM to 3mM)
and double the expected extension time
3mM Mg2+, 3:00 extension (bottom right bands)
Lower magnesium in other lanes
51 191 178 70 54
2mM Mg2+, 1:45 extension(I11V, I51V, I127V, I152V)
3mM Mg2+, 3:00 extension with new primers
55 93 55 93 55 93
PfuUltra II QuickChange Errors Primer sequence repeated upstream of mutation/annealing site
Sequence data from an I51V transformant colony (top) and an I193V transformant colony (bottom)
PfuUltra II Separated Primers Mutagenesis
Separate primers into two individual PCRs
No success
Q5 QuickChange Mutagenesis New Q5 polymerase in 2X master mix
Success for 7 mutations
Failure for 4 mutations (sequencing errors/no transformants)
191 187 184 178 176 70 51
Q5 Overlapping Primers Use overlapping primers
Exponential amplification
Can bridge nicks in newly synthesized DNA
Transformants for remaining 4 mutations. 1 sequence confirmed, 3 TBD
Liu H. and Naismith J.H. (2008)
Protein Prep Grow transformed C3013s in deuterium oxide minimal media
Add precursors and induce
Cobalt ion affinity chromatography
Future: Search for a Drug Finish purifying mutant protein and assigning NMR peaks
Go through chemical library
Compare NMR spectrums with wild-type to identify ligand binding
Synthesize allosteric inhibitor
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