Site Directed Mutagenesis of Protein PurE

Megan SilasFrom the University of Illinois at Urbana

ChampaignIn Dr. Fung’s Lab in the Department of

Chemistry

Outline Project Overview

Bacillus anthracis Purines PurE

Experimental Procedures and Results Primer Design Polymerase Chain Reaction Transformation Sequencing Protein Purification Activity Assay

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. <http://www.uic.edu/htbin/cgiwrap/bin/uicnews/articledetail.cgi?id=15363>.

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 moreSamant, 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 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

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 ribonucleotideNCAIR: N5-carboxyamino-imidizole ribonucleotideCAIR: 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

Certain amino acid residues are highly conserved

Critical to function and present in the active siteMathews, 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

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 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

Primer Design cDNA for baPurE

Primer Design:GGT GGA GCA GCG AAT TTA CCG GGA ATG

CAT = codon for HistidineAAT = 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

DNA Isolation Use DH5α cells containing a plasmid with baPurE

cDNA

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

DNA Gel Electrophoresis

To determine whether PCR was successful

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 DH5α cells

Successful PCR Results:

Transformation

Process of inserting a plasmid into competent cells DH5α 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

DNA Sequencing

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 4Analyzed results using: http://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html a feature available through the European Bioinformatics Institute

Sequencing ResultsColony 3

Primer

Mutation from CAT to AAT

Things to notice: All other nucleotides

are identical No insert

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

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

LB+amp liquid media Induce cells with Isopropyl β-D-1-

thiogalactopyranoside (IPTG) 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 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

" Affinity Chromatography." Wikipedia, the Free Encyclopedia. Web. 26 July 2011. http://en.wikipedia.org/wiki/Affinity_chromatography. .

GST = glutathione-S-transferase

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

Activity Assay Results

CA

IR (

A26

0)

Time (minutes)

Enzyme ΔA260/min

Specific Activity (μmolmin-1mg-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

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

Acknowledgements The financial support from the

National Science FoundationEEC-NSF Grant # 1062943

Dr. Fung, Nina Wolf, and Esther Ng REU Program Facilitators:

Dr. Takoudis, Dr. Jursich, and Arman Butt