Proteins are diverse polymers of covalently linked amino acids. They play a role in almost every biological process that occurs within an organism. Twenty different amino acids are genetically encoded by mammalian cells to build proteins. The sequence of these amino acids determines the protein’s final shape, structure, and function. Modern molecular cloning techniques allow for the genetic encoding and expression of mutant proteins that have one or more amino acids replaced with one of the others. The roles of individual amino acids in a protein can therefore be studied. Proteins with novel functions have also been designed or evolved using this technology. However, the genetic code is limited to the twenty natural amino acids.
Nonnatural amino acids have unique side groups that not found on any of the twenty natural amino acids. They can be site-specifically incorporated using a mutant orthogonal suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair. Each pair only allows for one type of nonnatural amino acid to be genetically encoded. This technology has resulted in the incorporation of over fifty different types of nonnatural amino acids into proteins in prokaryotic and eukaryotic cells. Unfortunately, most of these pairs are not orthogonal outside of prokaryotic systems and only a few have been developed for mammalian cells. To create more mammalian pairs a nonnatural aaRS has to be evolved and screened in a cumbersome process. In this dissertation an approach is outlined that can be used to change the orthogonality of existing nonnatural suppressor tRNA/aaRS pairs. As a result of the orthogonality change many previously unavailable pairs can be shuttled into mammalian cells. The ability to genetically encode a 21st amino acid is a powerful tool in the study and engineering of proteins. / text
| Identifer | oai:union.ndltd.org:UTEXAS/oai:repositories.lib.utexas.edu:2152/ETD-UT-2011-08-4095 |
| Date | 15 January 2013 |
| Creators | Xiang, Liang |
| Source Sets | University of Texas |
| Language | English |
| Detected Language | English |
| Type | thesis |
| Format | application/pdf |
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