Dual Noncanonical Amino Acid Incorporation Enabling Chemoselective Protein Modification at Two Distinct Sites in Yeast

Abstract

Proteins containing noncanonical amino acids (ncAAs) provide opportunities for dissecting fundamental biological processes and engineering protein therapeutics with diverse chemistries. Incorporation of more than one ncAA into a single construct can endow a protein with multiple useful features, such as unique molecular recognition and covalent crosslinking. Herein, for the first time, we encode two chemically distinct ncAAs into proteins prepared in Saccharomyces cerevisiae. To complement ncAA incorporation in response to the amber (TAG) stop codon in yeast, we evaluated opal (TGA) stop codon suppression using three distinct orthogonal translation systems (OTSs): E. coli tyrosyl-tRNA synthetase/tRNATyr, E. coli leucyl-tRNA synthetase/tRNALeu, and M. alvus pyrrolysyl-tRNA synthetase/tRNAPyl. We observed selective TGA readthrough without detectable cross-reactivity from host translation components. Further, we found that multiple factors impacted TGA readthrough efficiency in a manner analogous to TAG readthrough, including the local nucleotide context surrounding stop codons and deletion of selected genes from the yeast genome. In contrast, the identity of the suppressor tRNA affected TGA readthrough efficiency quite differently than TAG readthrough efficiency. Altogether, these observations facilitated the systematic investigation of dual ncAA incorporation in response to TAG and TGA codons within both intracellular and yeast-displayed protein constructs. Employing a proficient combination of OTSs, pairs of ncAAs could be encoded within both protein formats with efficiencies up to 6% of wildtype protein controls. Moreover, exploration of dual ncAA incorporation in yeast display format aided in demonstrating important applications of doubly-substituted proteins. First, dual ncAA incorporation within a simple synthetic antibody construct resulted in retention of antigen binding functionality, indicating that the doubly-substituted proteins can retain their basic functions. Second, presentation of reactive alkyne and azide functionalities within yeast-displayed constructs enabled sequential installation of two distinct chemical probes using bioorthogonal reactions--strain-promoted- and copper-catalyzed azide-alkyne cycloadditions (SPAAC and CuAAC)--on the yeast surface. This confirmed the ability to chemoselectively modify yeast-displayed proteins at two distinct sites. Furthermore, by utilizing a soluble form of a doubly substituted construct, we validated the feasibility to selectively double label proteins in solution in a single pot reaction. Overall, our work facilitates the addition of a 22nd amino acid to the yeast genetic code and provides an important toolkit that expands the scope of applications of ncAAs for basic biological research and drug discovery in yeast.

The Evolving Landscape of Antibody–Drug Conjugates: In Depth Analysis of Recent Research Progress

“…Finally, suppression of two of the three stop codons allows peptides to be translated with two NCAA, which allows two different warheads to be attached to a single ADC. 230 The difficulty of incorporating NCAA into an antibody likely means that the use of NCAA-based conjugation reduces the possible drug capacity of the ADC.…”