Site-Specific Incorporation of Two ncAAs for Two-Color Bioorthogonal Labeling and Crosslinking of Proteins on Live Mammalian Cells

Cell Rep. 2020 Jun 23;31(12):107811. doi: 10.1016/j.celrep.2020.107811.

Abstract

The pyrrolysyl-tRNA/pyrrolysyl-tRNA synthetase (PylT/RS) pair from the archaeon Methanosarcina mazei (Mma) is widely used in protein engineering to site-specifically introduce noncanonical amino acids (ncAAs) through nonsense codon suppression. Here, we engineer the PylT/RS pair encoded by Methanogenic archaeon ISO4-G1 (G1) to be orthogonal to Mma PylT/RS and alter the G1 PylRS active site to accept a complementary ncAA spectrum. We combine the resulting mutual orthogonal pairs for site-specific dual ncAA incorporation of two lysine analogs with high selectivity and efficiency. Demonstrating the robustness of the system, we incorporate two ncAAs with compatible bioorthogonal reactivity into a Notch receptor, as well as a G protein-coupled receptor. We show that selective and site-specific incorporation of two ncAAs allows for two-color bioorthogonal labeling as well as chemical-controlled crosslinking of surface proteins on live mammalian cells.

Keywords: Pyrrolysyl-tRNA-synthetase; amber suppression; bioorthogonal labeling; cell surface receptors; fluorescence microscopy; fluorescent labeling; genetic code expansion; noncanonical amino acids; stop codon suppression; unnatural amino acid mutagenesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / metabolism*
  • Amino Acyl-tRNA Synthetases / metabolism
  • Animals
  • Base Sequence
  • Cell Line
  • Cell Survival
  • Cross-Linking Reagents / chemistry*
  • HEK293 Cells
  • Humans
  • Lysine / metabolism
  • Mammals / metabolism*
  • Methanosarcina / metabolism
  • Nucleic Acid Conformation
  • Proteins / metabolism*
  • RNA, Transfer / chemistry
  • RNA, Transfer / genetics
  • Receptors, Cell Surface / metabolism
  • Receptors, G-Protein-Coupled / metabolism
  • Staining and Labeling*
  • Substrate Specificity

Substances

  • Amino Acids
  • Cross-Linking Reagents
  • Proteins
  • Receptors, Cell Surface
  • Receptors, G-Protein-Coupled
  • RNA, Transfer
  • Amino Acyl-tRNA Synthetases
  • Lysine