N-terminal labeling of proteins using initiator tRNA

Methods. 2005 Jul;36(3):252-60. doi: 10.1016/j.ymeth.2005.04.003.


Methodology based on tRNA mediated protein engineering is described for the introduction of fluorophores and other labels at the N-terminus of proteins produced in cell-free translation systems. One method for low-level (trace) N-terminal labeling is based on the use of an Escherichia coli initiator tRNA(fMet) misaminoacylated with methionine modified at the alpha-amino group. In addition to the normal formyl group, the protein translational machinery incorporates the fluorophore BODIPY-FL and the affinity tag biotin at an N-terminal end of the nascent protein. A second method for higher N-terminal labeling uses a chemically aminoacylated amber initiator suppressor tRNA and a DNA template which contains a complementary amber (UAG) codon instead of the normal initiation (AUG) codon. This more versatile approach is demonstrated using a variety of N-terminal markers including fluorescein, biotin, PC-biotin, and a novel dual marker conjugate (Biotin/BODIPY-FL).

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Escherichia coli / genetics
  • Fluorescent Dyes / chemistry
  • Protein Biosynthesis*
  • Protein Engineering / methods*
  • Proteins / chemistry*
  • RNA, Transfer / chemistry
  • RNA, Transfer, Met / chemistry*
  • Transfer RNA Aminoacylation


  • Fluorescent Dyes
  • Proteins
  • RNA, Transfer, Met
  • RNA, Transfer