Expanding the Scope of Multisite Noncanonical Amino Acid Mutagenesis

Abstract

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.