Transcript Megan Silas - University of Illinois at Chicago

Site Directed Mutagenesis of
Protein PurE
Megan Silas
From the University of Illinois at Urbana Champaign
In Dr. Fung’s Lab in the Department of Chemistry
Outline
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Project Overview
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Bacillus anthracis
Purines
PurE
Experimental Procedures and Results
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Primer Design
Polymerase Chain Reaction
Transformation
Sequencing
Protein Purification
Activity Assay
Bacillus anthracis – Anthrax
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A risk to national security, biological warfare
Fatal when untreated
Routes of entry to the body:
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Absorption through skin
Inhalation
Ingestion and then absorption through the digestive
tract
Need a novel antibiotic to target bacteria that are
resistant to current drugs
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How can we exploit current knowledge to help discover
alternative treatments?
Hostettler, Sam. "$14M Project to Develop Antibiotics against Biowarfare." UIC News. University of Illinois at Chicago, 18 May 2011. Web. 06 June 2011.
<http://www.uic.edu/htbin/cgiwrap/bin/uicnews/articledetail.cgi?id=15363>.
Bacteria
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In order to survive in human plasma, bacteria
must rely on de novo synthesis of many
different molecules
Studies show nucleotide (purine and pyrimidine)
biosynthesis to be the most critical
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Limited availability of nucleotides in human blood
Purines:
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A major component of DNA, RNA, ATP, GTP, and more
Samant, Shalaka, Hyunwoo Lee, Mahmood Ghassemi, Juan Chen, James L. Cook, Alexander S. Mankin, and Alexander A. Neyfakh. "Nucleotide Biosynthesis Is Critical for Growth of
Bacteria in Human Blood." PLoS Pathogens 4.2 (2008): E37.
"Purine." Wikipedia, the Free Encyclopedia. Web. 06 June 2011. http://en.wikipedia.org/wiki/Purine.
Purine Synthesis
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De novo synthesis of purines requires many different enzymes
Zhang, Y., M. Morar, and S. E. Ealick. "Structural Biology of the Purine Biosynthetic Pathway." Cellular and Molecular Life Sciences 65.23 (2008): 3699-724
PurE
N5-Carboxyaminoimidazole ribonucleotide mutase
N5-CAIR mutase
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Vertebrates
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Use PurE (Class II)
Unique mechanism to
convert from AIR to CAIR
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Bacteria
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Use combination of PurK and PurE
PurK creates NCAIR
NCAIR is converted to CAIR in a reversible
reaction catalyzed by PurE (Class I)
AIR: 5-aminoimidazole ribonucleotide
NCAIR: N5-carboxyamino-imidizole ribonucleotide
CAIR: 4-carboxy-5-aminoimidazole ribonucleotide
Zhang, Y., M. Morar, and S. E. Ealick. "Structural Biology of the Purine Biosynthetic Pathway." Cellular and Molecular Life Sciences 65.23 (2008): 3699-724
PurE
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Certain amino
acid residues are
highly conserved
Critical to function
and present in
the active site
Mathews, Irimpan I., T. Joseph Kappock, JoAnne Stubbe, and Steven E. Ealick. "Crystal Structure of Escherichia Coli PurE, an Unusual Mutase in the Purine Biosynthetic
Pathway." Structure 7.11 (1999): 1395-406.
Image: PDB Files 1XMP (yellow) and 1D7A (green), superimposed by N. Wolf in Dr. Fung’s Lab
baPurE
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In the PurE enzyme of
B. anthracis (baPurE),
one of these residues is
Histidine (H) 70
My project involves
mutating this residue
to Argenine (N)
H70N
Site Directed Mutagenesis
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Changing an amino acid residue of interest.
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Alter the structure of a protein
Determine effect on functionality
Primer: a complementary oligonucleotide (approx.
18-27 base pairs) with a point mutation at the
center such that the new codon will change the
single amino acid of interest
Primer Design
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cDNA for baPurE
ATG
GAA
CCG
ATG
ATT
GCA
AAA
CCA
TCA
CAT
GAA
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AAA TCA CTA GTT GGA GTC ATA ATG GGA AGC ACG TCA GAC TGG
ACA ATG AAA TAT GCT TGT GAC ATT TTA GAT GAA TTA AAT ATA
TAT GAG AAA AAG GTT GTA TCC GCT CAT CGG ACT CCG GAT TAT
TTT GAA TAT GCA GAG ACG GCT CGT GAA CGT GGA TTG AAA GTT
ATT GCT GGA GCT GGT GGA GCA GCG CAT TTA CCA GGA ATG GTT
GCG AAG ACG AAT CTT CCT GTA ATC GGA GTT CCA GTT CAA TCA
GCG TTA AAC GGC TTA GAT TCA TTA TTA TCC ATC GTC CAA ATG
GGA GGG GTT CCA GTT GCA ACT GTT GCA ATT GGT AAG GCT GGT
ACA AAT GCT GGT TTA CTT GCT GCA CAA ATA CTT GGA TCA TTC
GAT GAC ATA CAT GAT GCA TTA GAA TTG AGA AGA GAA GCA ATT
AAA GAT GTG CGC GAA GGT AGT GAG CTA GTA TGA
Primer Design:
GGT GGA GCA GCG AAT TTA CCG GGA ATG
CAT = codon for Histidine
AAT = codon for Argenine
DNA Isolation
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Use DH5α cells containing a plasmid with baPurE cDNA
Polymerase Chain Reaction
(PCR)
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Used to amplify short fragments
of DNA without using cells
Introduce primer to the plasmid
containing the wild type cDNA
Complementary regions will
anneal
Elongation will create a new
plasmid containing the desired
mutation that was initially
present in the primer
DNA Gel Electrophoresis
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To determine whether PCR was successful
Why is my PCR not working?
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Multiple unsuccessful PCRs:
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Varying cycling temperatures
Varying concentrations for template, primers and
dNTPs
Varying polymerase (“hot start” and pfu)
Potential problem with the template?
Re-isolate DNA from DH5α cells
Successful PCR Results:
Transformation
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Process of inserting a plasmid into
competent cells
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DH5α cells are engineered to be exceptional at
accepting foreign plasmids and replicating those
plasmids – competent cells
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Cells must have Ampicillin resistance
to grow on LB-amp plate
Growth implies a successful
transformation
No growth on the negative plate
confirms effectiveness of Ampicillin
DNA Sequencing
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Grew cultures of four distinct colonies in four
different 4 mL LB+amp liquid media
Extracted DNA from all colonies and sent for
sequencing at the Research Resources
Center facilities available at UIC
DNA Sequencing Results
Colony 1, 2, & 4
1
2
Analyzed results using: http://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html a feature available through the European Bioinformatics Institute
4
Sequencing Results
Colony 3
Mutation from CAT to AAT
Primer
Things to notice:
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All other nucleotides are
identical
No insert
Protein Purification
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Use DNA from:
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Colony 3 cells to create H70N protein
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Protein will have an identical amino acid sequence as
the wild type PurE, except for Arginine at position 70
Determine subsequent change in functionality
Colony 1 cells to create a truncated protein
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The insert contains a stop codon
Protein only has 98 amino acids instead of 161
Removing part of the active site
Predict no functionality
Creating H70N & Truncated PurE
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Transform DNA into BL21 cells
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Grow BL21 cells in two flasks of 2 L LB+amp
liquid media
Induce cells with Isopropyl β-D-1thiogalactopyranoside (IPTG)
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BL21 Cells are a mutated form of E. coli that over
produce proteins
Increases protein production and is not metabolized
by cells
Freeze cells overnight in -80ºC
" Isopropyl β-D-1-thiogalactopyranoside." Wikipedia, the Free Encyclopedia. Web. 26 July 2011. http://en.wikipedia.org/wiki/Isopropyl_%CE%B2-D-1-thiogalactopyranoside.
Protein Purification
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Affinity Column Chromatography
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PurE is a GST-fusion protein
GST binds to glutathione resin column
Can be released using elution buffer
Use proteolytic enzyme, thrombin,
to cut PurE from GST
Columns used to separate PurE
GST = glutathione-S-transferase
" Affinity Chromatography." Wikipedia, the Free Encyclopedia. Web. 26 July 2011. http://en.wikipedia.org/wiki/Affinity_chromatography. .
Activity Assay
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Use CAIR as reactant
CAIR will disappear
as it is converted into
NCAIR by PurE
Measure change in
absorbance due to disappearance of CAIR
Compare rate of reaction catalyzed by WT
PurE versus H70N and truncated PurE
Meyer, E., N.J. Leonard, B. Bhat, J. Stubbe, and J.M. Smith. "Purification and characterization of the purE, purK, and purC gene products: identification of a previously unrecognized energy
requirement in the purine biosynthetic pathway.” Biochemistry 31.21 (1992): 3699-724
CAIR (A260)
Activity Assay Results
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Time (minutes)
Enzyme
ΔA260/min Specific Activity
WT PurE
-0.0162
8.5
H70N
-0.0013
0.7
Truncate
d
0
0.3
(μmol min-1 mg-1)
Specific Activity:
 How much reactant is
converted to product
per minute per
milligram of enzyme
Conclusion
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Accomplishments
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Designed an ideal primer for the H70N mutation
Used PCR to obtain recombinant DNA with H70N mutation
Created DNA coding for a truncated PurE enzyme
Transformed the DNA into BL21 cells
Prepared H70N and truncated proteins
Determined enzymatic activity of these proteins
Acknowledgements
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The financial support from the National
Science Foundation
EEC-NSF Grant # 1062943
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Dr. Fung, Nina Wolf, and Esther Ng
REU Program Facilitators:
Dr. Takoudis, Dr. Jursich, and Arman Butt