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Site Directed Mutagenesis of Protein PurE

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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 – PowerPoint PPT presentation

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Title: Site Directed Mutagenesis of Protein PurE


1
Site Directed Mutagenesis of Protein PurE
  • Megan Silas
  • From the University of Illinois at Urbana
    Champaign
  • In Dr. Fungs Lab in the Department of Chemistry

2
Outline
  • Project Overview
  • Bacillus anthracis
  • Purines
  • PurE
  • Experimental Procedures and Results
  • Primer Design
  • Polymerase Chain Reaction
  • Transformation
  • Sequencing
  • Protein Purification
  • Activity Assay

3
Bacillus anthracis Anthrax
  • A risk to national security, biological warfare
  • Fatal when untreated
  • Routes of entry to the body
  • 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
  • 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. lthttp//www.uic.edu/htbin/cgiwr
ap/bin/uicnews/articledetail.cgi?id15363gt.
4
Bacteria
  • 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
  • Limited availability of nucleotides in human
    blood
  • Purines
  • 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.
5
Purine Synthesis
  • 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
6
PurEN5-Carboxyaminoimidazole ribonucleotide
mutaseN5-CAIR mutase
  • Vertebrates
  • Use PurE (Class II)
  • Unique mechanism to convert from AIR to CAIR
  • Bacteria
  • 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
7
PurE
  • 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.
Fungs Lab
8
baPurE
  • 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

9
Site Directed Mutagenesis
  • Changing an amino acid residue of interest.
  • 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

10
Primer Design
  • cDNA for baPurE
  • Primer Design
  • GGT GGA GCA GCG AAT TTA CCG GGA ATG
  • CAT codon for Histidine
  • AAT codon for Argenine

ATG AAA TCA CTA GTT GGA GTC ATA ATG GGA
AGC ACG TCA GAC TGG GAA ACA ATG AAA TAT
GCT TGT GAC ATT TTA GAT GAA TTA AAT ATA
CCG TAT GAG AAA AAG GTT GTA TCC GCT CAT
CGG ACT CCG GAT TAT ATG TTT GAA TAT GCA
GAG ACG GCT CGT GAA CGT GGA TTG AAA GTT
ATT ATT GCT GGA GCT GGT GGA GCA GCG CAT
TTA CCA GGA ATG GTT GCA GCG AAG ACG AAT
CTT CCT GTA ATC GGA GTT CCA GTT CAA TCA
AAA GCG TTA AAC GGC TTA GAT TCA TTA TTA
TCC ATC GTC CAA ATG CCA GGA GGG GTT CCA
GTT GCA ACT GTT GCA ATT GGT AAG GCT GGT
TCA ACA AAT GCT GGT TTA CTT GCT GCA CAA
ATA CTT GGA TCA TTC CAT GAT GAC ATA CAT
GAT GCA TTA GAA TTG AGA AGA GAA GCA ATT
GAA AAA GAT GTG CGC GAA GGT AGT GAG CTA
GTA TGA
11
DNA Isolation
  • Use DH5a cells containing a plasmid with baPurE
    cDNA

12
Polymerase Chain Reaction (PCR)
  • 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

13
DNA Gel Electrophoresis
  • To determine whether PCR was successful

14
Why is my PCR not working?
  • Multiple unsuccessful PCRs
  • 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 DH5a cells

15
Successful PCR Results
16
Transformation
  • Process of inserting a plasmid into competent
    cells
  • DH5a cells are engineered to be exceptional at
    accepting foreign plasmids and replicating those
    plasmids competent cells
  • 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

17
DNA Sequencing
  • Grew cultures of four distinct colonies in four
    different 4 mL LBamp liquid media
  • Extracted DNA from all colonies and sent for
    sequencing at the Research Resources Center
    facilities available at UIC

18
DNA Sequencing Results Colony 1, 2, 4
1
2
4
Analyzed results using http//www.ebi.ac.uk/Tools
/psa/emboss_needle/nucleotide.html a feature
available through the European Bioinformatics
Institute
19
Sequencing ResultsColony 3
Mutation from CAT to AAT
Primer
Things to notice
  • All other nucleotides are identical
  • No insert

20
Protein Purification
  • Use DNA from
  • Colony 3 cells to create H70N protein
  • 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
  • The insert contains a stop codon
  • Protein only has 98 amino acids instead of 161
  • Removing part of the active site
  • Predict no functionality

21
Creating H70N Truncated PurE
  • Transform DNA into BL21 cells
  • BL21 Cells are a mutated form of E. coli that
    over produce proteins
  • Grow BL21 cells in two flasks of 2 L LBamp
    liquid media
  • Induce cells with Isopropyl ß-D-1-thiogalactopyran
    oside (IPTG)
  • Increases protein production and is not
    metabolized by cells
  • Freeze cells overnight in -80ºC

" Isopropyl ß-D-1-thiogalactopyranoside."
Wikipedia, the Free Encyclopedia. Web. 26 July
2011. http//en.wikipedia.org/wiki/Isopropyl_CEB
2-D-1-thiogalactopyranoside.
22
Protein Purification
  • Affinity Column Chromatography
  • 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_chromatograp
hy. .
23
Activity Assay
  • 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
24
Activity Assay Results
Enzyme ?A260/min Specific Activity (µmol?min-1?mg-1)
WT PurE -0.0162 8.5
H70N -0.0013 0.7
Truncated 0 0.3
  • Specific Activity
  • How much reactant is converted to product per
    minute per milligram of enzyme

25
Conclusion
  • Accomplishments
  • 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

26
Acknowledgements
  • The financial support from the National Science
    Foundation
  • EEC-NSF Grant 1062943
  • Dr. Fung, Nina Wolf, and Esther Ng
  • REU Program Facilitators Dr. Takoudis, Dr.
    Jursich, and Arman Butt
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