Improved Eukaryotic Genetic Code Expansion

Abstract

Expanding the genetic code allows for incorporating amino acids not found in nature into an already extensive and constantly expanding library of known proteins. These non-standard amino acids can bring new functionality to proteins, allow for enhanced research of proteins, and keep genetically engineered organisms biologically contained in the environment. Most research has sought to allow for the possibility of incorporating a non-standard amino acid into a protein of interest, but not the specificity of this incorporation. Without recoding the entire genome there are hundreds or thousands of incorporation sites in a given organism even when using the most rare codons, such as the amber stop codon used in most recoding and non-standard amino acid incorporation methods. I created an approach to promote efficient non-standard amino acid incorporation into a protein of interest in non-recoded budding yeast Saccharomyces cerevisiae by including a cleavable n-terminal sequence that encourages specific incorporation of a nonstandard amino acid into a specified protein. During my dissertation research I attempted to inhibit factors associated with premature stop codon stalling on translating ribosomes. These factors are known to destabilize mRNAs that are recognized as containing premature stop codons, and by locally inhibiting these factors on the translating ribosome I observed increased non-standard amino acid incorporation and specificity of incorporation. Additionally, I attempted to characterize the mode of action of this inhibition and the efficacy compared to traditional approaches of non-standard amino acid incorporation. Increased efficiency and specificity of incorporation will allow for the use of hundreds of chemical structures developed in the past few decades which were previously unusable for research and manufacturing purposes due to high background without removing all competing codons through recoding, a feat which has not been achieved in Eukaryotes to date.