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Master of Science (MS)
Antos, John M.
Anthony-Cahill, Spencer J.
Spiegel, P. Clint
The versatility of sortase-mediated ligations as a protein modification technique has been well demonstrated, but the efficiency of these reactions suffers from inherent reversibility. Solutions to this issue have been reported, however these methods are accompanied by additional limitations of the sortase-mediated ligation (SML) strategy. A preferable methodology would include the smallest possible modification site without restricting the point of ligation. One promising solution to this issue is the expansion of the LPXTG SrtA recognition sequence to LPXTGGH, giving the excised fragment an N-terminal GGH motif. This minor alteration has been shown to allow complexation of the excised fragment with Ni2+ ions, thus sequestering this component from the reaction and improving yields through hindered reversibility. In this thesis, we explore the scope of this metal-assisted sortase-mediated ligation (MASML) approach, including the Ni2+-enhanced modification of full sized proteins with a number of useful chromophores. Furthermore, this approach was shown to be compatible with the installation of PEG and a cyclooctyne bioorthogonal ligation handle. In total, this work demonstrates that MASML is compatible with a range of high value protein targets and modifications, and shows how MASML is a straightforward method for improving the efficiency of sortase-mediated protein engineering strategies.
Western Washington University
Copying of this thesis in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this thesis for commercial purposes, or for financial gain, shall not be allowed without the author's written permission.
Reed, Sierra, "Driving Sortase-Mediated Ligations Using Metal-Coordinating Peptides" (2018). WWU Graduate School Collection. 700.
Available for download on Saturday, June 29, 2019