Incorporation of Unnatural Amino Acids into Proteins Expressed in Mammalian Cells

Abstract

The site-specific incorporation of unnatural amino acids (Uaas) via genetic code expansion provides a powerful method to introduce synthetic moieties into specific positions of a protein directly in the live cell. The technique, first developed in bacteria, is nowadays widely applicable in mammalian cells. In general, different Uaas are incorporated with different efficiency. By comparing the incorporation efficiency of several Uaas recently designed for bioorthogonal chemistry, we present here a facile dual-fluorescence assay to evaluate relative yields of Uaa incorporation. Several biological questions can be addressed using Uaas tools. In recent years, photo-cross-linking Uaas have been extensively applied to map ligand-binding sites on G protein-coupled receptors (GPCRs). We describe a simple and efficient two-plasmid system to incorporate a photoactivatable Uaa into a class B GPCR, and demonstrate cross-linking to its nonmodified natural ligand.

Keywords: Corticotropin-releasing factor receptor; Dual-fluorescence; Expanded genetic code; Fluorescence assay; GPCRs; Incorporation of unnatural amino acids; Mammalian cells; Photo-cross-linking; PylRS; Two-plasmid system; Uaas for bioorthogonal chemistry.