Date of Graduation

Summer 2018

Degree

Master of Science in Chemistry

Department

Chemistry

Committee Chair

Keiichi Yoshimatsu

Keywords

spycatcher, spytag, kinetics, fluorescence polarization, isopeptide bond

Subject Categories

Analytical Chemistry | Biochemistry | Biotechnology

Abstract

Autocatalytic intramolecular isopeptide bonds have been found in nature in certain gram-positive bacterial pilus structures. Recently, splitting of these domains that are capable of autocatalytic intramolecular isopeptide bond formation have been applied to create stable, selective, bio-orthogonal Catcher/Tag systems. The CnaB2 domain found in the FbaB pilus structure of Streptococcus pyogenes, has yielded the Catcher/Tag, Protein/Peptide systems termed SpyCatchter and SpyTag. Recent study has focused on tag optimization, stability and bio-orthogonality evaluation, along with applications in bioconjugation. I have recombinantly expressed SpyCatcher and SpyTag-fused proteins in E.coli, and conjugated them to fluorescent probes in order for use in fluorescence polarization/depolarization study. Using this system, I have observed the dependence of SpyTag concentration on the formation of the isopeptide bond. I have also been able to track the formation of this bond in real time.

Copyright

© Samuel Patricc Kasson

Available for download on Saturday, August 01, 2020

Open Access

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