Thesis advisor: Abhishek Chatterjee / Noncanonical amino acid (ncAA) mutagenesis provides powerful new ways to probe and manipulate protein function both in vitro and in living cells. Increasing the number of ncAAs that can be site-specifically encoded can greatly expand the scope of this promising technology. We aimed to address the challenges that limit the multisite ncAA incorporation technology in both Escherichia coli and mammalian cells. Our work has significantly expanded the scope of this technology through the development of mutually compatible suppression systems and the optimization of their expression. Using these platforms, we further demonstrate powerful new applications of dual-ncAA incorporation technology both in E. coli and mammalian cells. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

With the objective to functionalize proteins for the understanding of their biological roles and developing protein-based biosensors, I have been developing methods to synthesize proteins with defined modifications and applying them to study protein functional roles and generate proteins with new properties. These methods rely on the read-through of an in-frame stop codon in mRNA by a nonsense suppressor tRNA specifically acylated with a noncanoncial amino acid (NAA) by a unique aminoacyl-tRNA synthetase and the genetic incorporation of this NAA at the stop codon site. NAAs either provide chemical handles for site-specific manipulation or mimic the posttranslational modifications, which are critical for understanding cellular regulations and signal transduction. The pyrrolysine synthetase (PylRS) has been wildly used to incorporate NAAs into proteins in E. coli. Taking advantage of PylRS, I have developed method to genetically incorporate ketone-containing N--acetyl-L-lysine analog, 2-amino-8-oxononanoic acid (KetoK), into proteins for their site-specific modifications and used it to mimic the protein lysine acetylation process. I have also modified the ribosome in order to improve the amber suppression efficiency and therefore to achieve incorporation of multiple copies of NAA into one protein. By overexpressing a truncated ribosomal protein, L11C, I have demonstrated 5-fold increase of amber suppression level in E. coli, leading to higher expression levels for proteins incorporated with NAAs. I have also demonstrated this method can be applied successfully to incorporate at least 3 NAAs into one protein in E. coli. With the success of incorporating multiple NAAs into one protein, I have further introduced two distinct NAAs into one protein simultaneously. This is done by using a wild type or evolved PylRS-pylTUUA pair and an evolved M. jannaschii tyrosyl-tRNA synthetase (MjTyrRS)-tRNACUA pair. By suppressing both UAG and UAA stop codons in one mRNA, a protein incorporated with two NAAs is synthesized with a decent yield. There is of great interest to incorporate new NAAs into proteins, which is done by library selection. By introducing both positive and negative selective markers into one plasmid, I have developed a one-plasmid selection method. In this method, the positive and negative selections are accomplished by in a single type of cells hosting a single selection plasmid.

Thesis advisor: Abhishek Chatterjee / Thesis advisor: Eranthie Weerapana / Over the past twenty years, the site-specific incorporation of unnatural amino acids (UAAs) into a target protein through genetic code expansion (GCE) has emerged as one of the foremost technologies to selectively modify proteins in their native cellular context. This technology relies on engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pairs that are orthogonal to the host cells’ endogenous aaRS/tRNA pairs. Traditionally, scientists look towards evolutionarily distant domains of life to identify orthogonal aaRS/tRNA pairs that can be further engineered for GCE applications in the host system. For example, bacterial aaRS/tRNA pairs are used for GCE in eukaryotes. The directed evolution of orthogonal aaRS/tRNA pairs for eukaryotic GCE has been less fortuitous due to the cumbersome nature of established yeast-based selection platforms. Recently, our lab circumvented this platform-based limitation by developing “altered translational machinery” (ATM) Escherichia coli strains that enabled the directed evolution of bacterial aaRS/tRNA pairs for eukaryotic GCE applications. In the ATM-tyrosyl (ATMY) E. coli strain, reintroduction of the E. coli tyrosyl-tRNA (tRNAEcTyrCUA) as a nonsense suppressor led to cross-reactivity with the endogenous E. coli glutaminyl-tRNA synthetase (EcGlnRS), restricting the activity range of aaRSs that could be selected, ultimately diminishing the scope of incorporable UAAs. To recover the dynamic range of this platform, cross-reactivity of the tRNAEcTyrCUA was eliminated through directed evolution of the tRNA acceptor stem. This new, orthogonal tRNA revealed weak mutant aaRSs whose suppression efficiencies were boosted through additional rounds of directed evolution. Improved aaRS mutants exhibited higher solubility, thermal stability, and suppression efficiency than their predecessor. While the newly engineered, orthogonal tRNAEcTyrCUA gave access to novel aaRS/tRNA pairs for eukaryotic GCE, some notable UAAs were still missing that could be incorporated with the archaeal Methanococcus jannaschii tyrosyl-tRNA synthetase (MjTyrRS)/tRNA pair in bacteria. Following a systematic investigation into the discrepancy between the E. coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNA and MjTyrRS/tRNA pairs, we found that it can be partially attributed to the low structural robustness of the EcTyrRS. This limitation was overcome by rationally designing chimeric TyrRSs composed of EcTyrRS and a structural homologue from the thermophilic bacterium Geobacillus stearothermophilus. The chimeric scaffolds demonstrated enhanced stability, activity, and resilience to destabilizing active site mutations, offering a potentially more attractive scaffold for GCE. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.