Evaluating Engineered MtPylRS Variants for Non-Standard Amino Acid Incorporation in HEK293 Cells

• Jacob Escobar, Chemical Engineering, University of Delaware

• Aditya Kunjapur, Chemical & Biomolecular Engineering, University of Delaware
• Kelvin Lee, Chemical & Biomolecular Engineering, University of Delaware

Genetic code expansion (GCE) enables the site-specific incorporation of non-standard amino acids (nsAAs) into proteins through the use of orthogonal translation systems (OTSs). Incorporation of nsAAs expands protein functionality for applications such as protein labeling, bioconjugation, and therapeutic protein engineering. Despite these applications, mammalian GCE remains limited by the efficiency of existing OTSs, resulting in relatively low nsAA incorporation efficiency. Here, we compare the parent Methanosarcina thermophila pyrrolysyl-tRNA synthetase (MtPylRS) with two engineered variants to determine whether these engineered mutations enhance nsAA incorporation efficiency.

HEK293 cells are transiently transfected with plasmids encoding the parent or engineered MtPylRS variants together with reporter and control constructs. Incorporation efficiency is then evaluated for three diverse nsAA chemistries. A dual-fluorescence reporter containing an in-frame UAG stop codon is used to quantify nsAA incorporation through amber stop-codon suppression, while a corresponding control construct lacking the UAG stop codon provides an upper bound for GFP expression. LSSmApple (RFP) reports overall protein expression, whereas TurboGFP reports successful nsAA incorporation. Relative Read-Through Efficiency (RRTE), calculated from normalized GFP and RFP fluorescence, enables quantitative comparison of incorporation efficiencies while accounting for differences in expression level. Because the two engineered MtPylRS variants are designed to increase affinity for nsAAs, they are expected to exhibit higher RRTE values than the parent MtPylRS system.

Identification of improved MtPylRS variants could advance mammalian genetic code expansion and broaden the use of nsAAs for biotechnology and therapeutic protein engineering. Future studies will evaluate the performance of optimized OTS variants in CHO cells and develop intracellular protein recovery methods for downstream characterization of nsAA-containing proteins.