RESEARCH OVERVIEW

Genetic code expansion is a powerful technique that enables the site-specific incorporation of non-standard amino acids into proteins. This is achieved using engineered tRNA and aminoacyl-tRNA synthetase pairs that function independently of the cell’s natural protein synthesis machinery. Unlike the 22 naturally occurring amino acids, non-standard amino acids offer diverse chemical functionalities, allowing researchers to create proteins with novel or enhanced properties. In my Ph.D. research, I leveraged genetic code expansion as a chemical biology tool to investigate fundamental biological questions.

Evolving aminoacyl-tRNA synthetases to incorporate D-amino acids

Proteins are almost exclusively composed of L-form amino acids, with only rare exceptions. By engineering the active site of the archaeal pyrolysyl-tRNA synthetase (PylRS), we have enabled the site-specific incorporation of D-form non-standard amino acids into proteins at amber codons. This work expands the chemical diversity available for protein engineering beyond the traditional L-form amino acid toolbox. In addition, modifying the N-terminal domain and linker region of PylRS has significantly improved the activity of the engineered enzyme. These enhancements address a key limitation in genetic code expansion by increasing the efficiency of modified synthetases, making the system more robust and practical for broader applications.

Synthetic Platform for Protein Post-Translational Modifications

Ubiquitination and SUMOylation are important post-translational modifications that regulate many cellular processes and are linked to serious human diseases. In particular, the dynamic interaction between ubiquitin and SUMO2 plays a critical role in DNA repair and replication. To better understand the biological significance of proteins modified with both ubiquitin and SUMO2, we developed a universal protein synthesis platform that enables site-specific attachment of these modifications. Unlike enzyme-based methods, which often produce mixtures with uncertain modification sites, our approach allows for the precise and uniform generation of ubiquitinated or SUMOylated proteins.

Tryptophan in Action: Probing Its Role at Enzyme Active Sites USP30 is a deubiquitinase located on the outer membrane of mitochondria, and it has been identified as a potential drug target for Parkinson's disease. Interestingly, we found that Trp475 is located near the active site of USP30 and may play a vital role in carrying out its function. To understand the role of Trp475 in more detail, we prepared five tryptophan analog-encoded USP30 variants with the help of an evolved pyrrolysyl-tRNA synthetase and tRNA pairs to examine their activity and selectivity toward di-ubiquitin. Our results suggest that Trp475 is critical in maintaining the activity and selectivity of USP30, and this finding may shed light on new drug designs in the future.

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