Honors College Thesis
 

Tuning tRNA and EF-Tu for the Efficient Site-specific Incorporation of Noncanonical Amino Acids into Proteins

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https://ir.library.oregonstate.edu/concern/honors_college_theses/pg15bn94c

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  • Genetic code expansion (GCE) is a technology that allows us to incorporate noncanonical amino acids (ncAAs) site-specifically into recombinant proteins using engineered translational machinery. Typically, an orthogonal aaRS/tRNA pair is engineered by directed evolution to recognize the ncAA and incorporate it at a reassigned codon while not interacting with the hosts endogenous translational machinery. Although optimization of the aaRS/tRNA pair is sufficient to make functional GCE systems for many ncAAs, the efficiency of ncAA translation is much lower than that of natural translation due to translational bottlenecks including non-optimal binding of ncAA-tRNA to the elongation factor Tu (EF-Tu). Here, I present a novel strategy to optimize the interaction between ncAA-tRNA and EF-Tu for any GCE system. Specifically, I describe a selection system in which both EF-Tu and tRNA variants are evolved in parallel for optimized amino acid binding pockets and T-stems. Using the fluorescent ncAA acridon-2-ylalanine as a test case, I show for the first time that co-evolving EF-Tu and EF-Tu/tRNA pairs is an effective strategy for improving the efficiency of ncAA translational systems at least 2-fold versus the progenitor system.
  • Key Words: Genetic code expansion, tRNA, T-stem, EF-Tu, directed evolution
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  • Ongoing Research
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  • 2021-06-02 to 2023-07-03

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