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.

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.

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.

FALL 2009 MASS SPEC. RESULTS

Fructose-bisphosphate aldolase confers no likely stabilization

Cystine transporter subunit also likely not stabilizing

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

METHODS

Concentrate Stage 4 Enzymes

SDS-PAGE => Coomassie => De-stain

In-gel trypsin digest

Mass spectroscopy: LC/MS-MS (ESI)

0.3 0.4 0.5 0.6 0.7 0.8 0.94.3

4.35

4.4

4.45

4.5

4.55

4.6

4.65

4.7

4.75

f(x) = − 0.546815760385734 x + 4.89575478608493

Rf

Log

(M

W)

MW DETERMINATION BY SDS-PAGE

Rf = band distance/dye front distance

RESULTS

RESULTSSample 1: 24.7 kDa

RESULTS Sample 2: 35.1 kDa

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.

REFERENCES Kurokawa, Yoichi, Hideki Yanagi, and Takashi Yura.

"Overexpression of Protein Disulfide Isomerase DsbC 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” The International 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>.