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Site Directed Mutagenesis of Protein PurE

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. Project Overview Bacillus anthracis Purines PurE Experimental Procedures and Results Primer Design

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Site Directed Mutagenesis of Protein PurE

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  1. 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

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

  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. 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) PurEN5-Carboxyaminoimidazole ribonucleotide mutaseN5-CAIR mutase 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. Fung’s 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 DH5α 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 DH5α cells

  15. Successful PCR Results:

  16. 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

  17. 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

  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 • All other nucleotides are identical • No insert Mutation from CAT to AAT Primer Things to notice:

  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 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.

  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_chromatography. .

  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 CAIR (A260) • Specific Activity: • How much reactant is converted to product per minute per milligram of enzyme Time (minutes)

  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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