Browsing by Subject "Protein Labeling"

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    Studies and applications of protein prenylation using isoprenoid analogues and site-directed mutagenesis
    (2014-06) Dozier, Jonathan Kramer
    The site-specific enzymatic modification of proteins offers a powerful tool both for the development of new research techniques and for the development of more effective therapeutics. Protein farnesyltransferase (PFTase) is a promising tool in the field of site-specific protein modification due to its substrate promiscuity, small recognition domain and mild reaction conditions. PFTase is an enzyme that attaches a farnesyl moiety, from farnesyl diphosphate (FPP), to a cysteine residue located at the C-terminus of proteins in a CaaX box consensus sequence. This dissertation seeks to used PFTase as a tool to develop new methods for both scientific research and biotechnologcical development. PFTase has been shown to utilize a number of non-natural FPP analogues that can be used to create covalent conjugates between proteins and other biologically relevant molecules. One of the main disadvantages of this approach, however, is that PFTase has a much lower activity when using FPP analogues that contain large non-natural functional moieties. We sought to develop and analyze different mutations of PFTase that increase the activity of the enzyme toward these FPP analogues. We found several PFTase mutants that can catalyze the addition of large FPP analogues to proteins at much faster rate than the wild-type enzyme. Additionally we showed that we could use PFTase to create a site-specific conjugate of Ciliary Neurotropic Factor (CNTF); a promising drug candidate for a number of different neurological conditions. In addition, we have begun analyzing its biological activity in vivo. This conjugate is a promising new development in the creation of protein therapeutics, that have a similar activity but a longer retention time in the body than previously drugs. Taken together this dissertation will demonstrate the value and utility of using PFTase for a number of different scientific applications.