2012 Summer Symposium Program - Middlebury College

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Matthew Thomas Stanley ‘15
Major: Undeclared
National Science Foundation
(Larrabee)
Jim Larrabee
William R. Kenan Jr. Professor
of Chemistry
Middlebury Summer Research Symposium 2012
that they facilitate the growth of S. mutans, then dietary carbohydrates will be
continuously fermented by this oral cariogen, leading to the accumulation of lactic
acid at the plaque-enamel interface. The end result is the deminerization of tooth
enamel and the onset of dental decay. A 25kDa SloR metalloregulatory protein was
described previously in the Spatafora laboratory as a modulator of various S. mutans
virulence attributes, including plaque architecture. A SloR knockout mutant, called
GMS584, as well as a SloR-overexpressing strain, called GMS541, were constructed
in the Spatafora laboratory and shown to have altered biofilm architectures on
scanning electron micrographs. As an obligate biofilm former, S. mutans is especially
resistant to antibiotics and antimicrobial agents that are commonly used to reduce
microbial load before dental manipulations and oral surgery. Chlorhexidine
digluconate is an antimicrobial that is commonly used in the clinical setting. In
this study, S. mutans UA159, GMS584, and GMS541 will be investigated in growth
determination, crystal violet release, and chlorhexidine susceptibility assays to
determine whether SloR has any impact on the access and susceptibility of S. mutans
biofilms to chlorhexidine digluconate. This research is significant because it can
reveal a novel role for SloR in antimicrobial susceptibility and to facilitate oral health
and hygiene practices.
Expression and purification of the enzyme
methionine aminopeptidase for magnetic circular
dichroism and equilibrium dialysis studies
Matthew Thomas Stanley and Jim Larrabee
Department of Chemistry and Biochemistry, Middlebury College, Middlebury VT 05753
The enzyme methionine aminopeptidase (MetAP) is a metallohydrolase that
facilitates the removal of the N-terminal methionine on select polypeptides. Interest
in this specific metallohydrolase stems from its significant role in angiogenesis
and its anti-bacterial implications. Production and purification of MetAP on a
large scale is a critical and time-consuming step, which provides the necessary
enzyme for later spectroscopic and thermodynamic metal-binding studies. This
work presents the specific procedure developed to express and purify MetAP using
Escherichia coli bacteria mutated to over-express the six-histidine tagged MetAP, the
protein was purified from the cells using a series of chromatographic methods. The
chromatographic method utilizes the histidine tag located on the protein by using a
nickel agarose gel to effectively separate the protein from other cell particulates. The
His-tag was cleaved, and the protein was purified further by using a mass exclusion
column. Successful isolation of MetAP was confirmed using an HPLC enzyme
activity assay. This procedure is robust and allows for a large yield of protein,
approximately 400 milligrams of protein from 25 grams of E. coli cells. Future
applications for this enzyme will include magnetic circular dichroism spectroscopy
and equilibrium dialysis studies of the cobalt-binding center to determine an
enzymatic mechanism for MetAP.