Honors College Thesis
 

Selection of an Orthogonal tRNA Synthetase for Trifluoromethyl-phenylalanine in Methanomethylophilus Alvus System

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https://ir.library.oregonstate.edu/concern/honors_college_theses/2j62sc875

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  • Genetic code expansion is a technology that allows for site-specific incorporation of noncanonical amino acids into recombinant proteins at amber stop codons using engineered translational machinery. The technology allows for expanded study into proteins, derived from the integration of novel chemical functions found in noncanonical amino acids, such as fluorescence, ligand binding and cross-linking. One such noncanonical amino acid, trifluoromethyl-phenylalanine, provides a strong signal for 19F Nuclear Magnetic Resonance spectroscopy. In order to incorporate trifluoromethyl-phenylalanine site specifically, orthogonal translational machinery must be developed. Specifically, an evolved pyrrolysine amino-acyl tRNA synthetase/tRNA pair must functionally incorporate trifluoromethyl-phenylalanine with high fidelity. The pair was selected from a library consisting of engineered pyrrolysine synthetases from the archaeal species Methanomethylophilus alvus. The system was chosen because the pyrrolysine synthetase is orthogonal to endogenous eukaryotic translational machinery. A selection process consisting of a positive selection, a negative selection, and a pALS2 screen was performed to find a synthetase that binds trifluoromethyl-phenylalanine with high efficiency and fidelity. The completed selection indicated that no engineered synthetase could bind trifluoromethyl-phenylalanine with the required efficiency or fidelity. There may be a lack of fidelity stemming from the size of the binding pocket being much larger than trifluoromethyl-phenylalanine combined with the non-canonical amino acid’s lack of unique electrostatic interactions. It is also possible the mutations required to bind trifluoromethyl-phenylalanine with high functionality and fidelity thermodynamically destabilize the synthetase, causing it to unfold and lose function.
  • Keywords: Fidelity, Fluorine Nuclear Magnetic Resonance spectroscopy, site-specific incorporation
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  • This work was supported in part by the GCE4All Biomedical Technology Development and Dissemination Center supported by National Institute of General Medical Science grant RM1-GM144227.
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