Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery

Abstract

Summary: Stop codon suppression using dedicated tRNA/aminoacyl-tRNA synthetase (aaRS) pairs allows for genetically encoded, site-specific incorporation of non-canonical amino acids (ncAAs) as chemical handles for protein labeling and modification. Here, we demonstrate that piggyBac-mediated genomic integration of archaeal pyrrolysine tRNA (tRNAPyl)/pyrrolysyl-tRNA synthetase (PylRS) or bacterial tRNA/aaRS pairs, using a modular plasmid design with multi-copy tRNA arrays, allows for homogeneous and efficient genetically encoded ncAA incorporation in diverse mammalian cell lines. We assess opportunities and limitations of using ncAAs for fluorescent labeling applications in stable cell lines. We explore suppression of ochre and opal stop codons and finally incorporate two distinct ncAAs with mutually orthogonal click chemistries for site-specific, dual-fluorophore labeling of a cell surface receptor on live mammalian cells. Motivation: Genetic code expansion via stop codon suppression is a powerful strategy to engineer proteins. Pyrrolysine tRNA (tRNAPyl)/pyrrolysyl-tRNA synthetase (PylRS) pairs from methanogenic archaea and engineered bacterial tRNA/aminoacyl-tRNA synthetase (aaRS) pairs are used for site-specific incorporation of non-canonical amino acids (ncAAs) in response to stop codons in mammalian cells. Routinely, ncAA incorporation is achieved by transient expression of the tRNA/aaRS pair leading to heterogeneous suppression. Genomic integration of tRNA/aaRS expression cassettes for more homogeneous, adjustable, and reproducible levels of protein, containing one or more ncAAs, will greatly benefit synthetic protein production, functional protein engineering, chemical control, and imaging applications in mammalian cells.

Keywords

• CP: Biotechnology
• CP: Molecular biology