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Emerging biotechnology to detect weak and/or transient protein-protein interactions

Protein-protein interactions are of great importance to a number of essential biological processes including cell cycle regulation, cell-cell interactions, DNA replication, transcription and translation. Thus, an understanding of protein-protein interactions is critical for understanding many facets of cell function. Unfortunately, the tools and methods currently in use to identify and study protein-protein interactions focus largely on high affinity, stable interactions. However, the majority of the protein-protein interactions involved in regulatory processes have weak affinities and are transient in nature. Therefore, it is important to develop new biotechnology capable of detecting weak and/or transient protein-protein interactions in vivo. Here, we describe four new methods that allow for the identification and study of weak and/or transient protein-protein interactions in vivo. First, we developed a rapid method to convert Escherichia coli orthogonal tRNA/synthetase pairs into an orthogonal system for mammalian cells in order to site-specifically incorporate unnatural amino acids into any gene of interest using stop codon suppression. This method will allow the expression and purification of proteins that carry normally transient post-translational modifications. Second, we successfully employed site-specific unnatural amino acid incorporation to chemically cross-link a known homodimer, Sortase A, in vivo. Third, we developed a novel tetracycline repressor-based mammalian two-hybrid system and successfully detected homo- and hetero-dimers that are known to have weak binding constants. Finally, a synthetic antibody (termed a synbody) that binds weakly to the SH3 domain of the proto-oncogene Abelson tyrosine kinase was developed. The synbody can potentially be used as a first generation drug and/or biomarker. We hope that the methods developed in this dissertation will enable the scientific community to better understand weak/transient protein-protein interactions in vivo. / text

Identiferoai:union.ndltd.org:UTEXAS/oai:repositories.lib.utexas.edu:2152/24366
Date30 April 2014
CreatorsThibodeaux, Gabrielle Nina
Source SetsUniversity of Texas
LanguageEnglish
Detected LanguageEnglish
TypeThesis
Formatelectronic
RightsCopyright is held by the author. Presentation of this material on the Libraries' web site by University Libraries, The University of Texas at Austin was made possible under a limited license grant from the author who has retained all copyrights in the works.

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