Examining the Activity of Sortase A Variants in Peptide Ligation

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Andrew Wood Pearl Tsang

Abstract

By Andrew Wood, Chemistry


Advisor: Pearl Tsang


Presentation ID: AM_D02


Abstract: Sortase A is an enzyme found in Gram positive bacteria. In those bacteria it catalyzes a reaction where a polypeptide chain is connected to the outer cell wall. This enzyme recognizes an LPXTG sequence on the substrate's polypeptide chain, where X denotes any amino acid, and catalyzes the attachment of this substrate to a second polypeptide chain beginning with at least one glycine. This reaction results in posttranslational protein ligation. Performing this ligation via enzymatic reaction can facilitate biophysical study of folded multidomain proteins under physiological conditions. In 2011, a penta-mutant of sortase A was created through directed evolution in order to increase the rate of reaction with primary substrate.�In order to assay the kinetic differences between these variants, we established a fluorescent peptide assay. Here, we report on the establishment of this assay as well as the varying reactivity of native enzyme and pentamutant, both as a transpeptidase and a hydrolase. Using High Pressure Liquid Chromatography (HPLC) we were able to observe peaks associated with the products and remaining reactants of our reactions. Gathered data from HPLC chromatograms and fluorescence indicate that the pentamutant sortase A is more active both as a transpeptidase and a protease.

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AM Poster Session -- Great Hall -- D: New Frontiers