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E. Coli Alkaline Phosphatase Heat Stability Continuation; the Search for a Suspected Chaperone

E. Coli Alkaline Phosphatase Heat Stability Continuation; the Search for a Suspected Chaperone. By: Desiree Morris, Thomas Yi, Angela Schlegel. Background. A group from spring 2011 ran a SDS PAGE gel on stage four AP. They found lower bands that could be DsbC .

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E. Coli Alkaline Phosphatase Heat Stability Continuation; the Search for a Suspected Chaperone

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  1. E. Coli Alkaline Phosphatase Heat Stability Continuation; the Search for a Suspected Chaperone By: Desiree Morris, Thomas Yi, Angela Schlegel

  2. Background • A group from spring 2011 ran a SDS PAGE gel on stage four AP. They found lower bands that could be DsbC. • When mass spec was run on the stage four enzyme, they found a protein that could be DsbC. • This could explain why pure enzyme from Sigma Aldrich is more thermally stable than stage four AP.

  3. Dsb • The Dsb family of proteins catalyze the formation of double bonds. • They are located in the periplasm of the E coli. • Kurokawa et al concluded that overexpression of DsbC stabilizes proteins with multiple double bonds. • DsbC is a dimeric protein with a monomeric MW of 23.3kDa • It fuctions as an isomerase and chaperone.

  4. Fall 2009 Mass Spec. Results • Fructose-bisphosphatealdolase confers no likely stabilization • Cystine transporter subunit also likely not stabilizing

  5. Goals • Determine identities of ~24.7 kDa and ~35.1 kDa proteins in the stage 4 sample Hypothesis • The lower molecular weight band is believed to be DsbC or one of the other Dsb proteins • The higher MW protein is predicted to have a role in stabilizing/forming disulfide bonds or another thermal stability function

  6. Methods Concentrate Stage 4 Enzymes SDS-PAGE => Coomassie => De-stain In-gel trypsin digest Mass spectroscopy: LC/MS-MS (ESI)

  7. MW Determination by SDS-PAGE Rf = band distance/dye front distance

  8. Results

  9. Results Sample 1: 24.7 kDa

  10. Results Sample 2: 35.1 kDa

  11. Future Experiments • Additional sample submission. • 2 bands were included in each sample. A greater amount needs to be added for better analysis • Run another gel. Larger Pore size for expansion of protein cluster in the 30.6 kDa region. Further investigation of the proteins through MS/MS • Why? DsbC is believed to be reoxidized by an uncharacterized protein acting as a disulfide isomerase (STRING). • Protein-protein interactions • Determine locations of protein interactions which could lead to a proposed method.

  12. References • Kurokawa, Yoichi, Hideki Yanagi, and Takashi Yura. "Overexpression of Protein Disulfide IsomeraseDsbC Stabilizes Multiple-Disulfide-Bonded Recombinant Protein Produced and Transported to the Periplasm in Escherichia Coli." Applied and Environmental Microbiology(2000) 66.9: 3960-3965. • Messens, Jori & Collet, Jean-François. “Pathways of Disulfide Formation in Escherichia coli” TheInternational Journal of Biochemistry & Cell Biology(2006) 38:1050-1062. • "STRING: Functional Protein Association Networks." STRING: Functional Protein Association Networks. Web. 24 Apr. 2012. <http://string-db.org/newstring_cgi/show_network_section.pl>.

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