Cell-free N-terminal protein labeling using initiator suppressor tRNA

Anal Biochem. 2004 Mar 1;326(1):25-32. doi: 10.1016/j.ab.2003.11.002.


A highly efficient method for the introduction of fluorophores and other markers at the N terminus of proteins produced in a cell-free extract has been developed. The method utilizes an amber (CUA) initiator suppressor tRNA chemically aminoacylated with a fluorophore-amino acid conjugate which is introduced into an Escherichia coli S30 cell-free translation system. The DNA template contains a complementary amber (UAG) codon instead of the normal initiation (AUG) codon. Using this approach, the fluorophore BODIPY-F1 (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a- diaza-s-indacene-3-propionic acid) has been incorporated at the N terminus of several model proteins. The specific labeling achieved (27-67%) using this approach is much higher than that of wild-type tRNAs. Several potential biophysical and biotechnological applications of this new technology are described.

Publication types

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

MeSH terms

  • Acylation
  • Amination
  • Boron Compounds
  • Calibration
  • Cell-Free System
  • Codon / genetics
  • Escherichia coli
  • Propionates / chemistry
  • Protein Biosynthesis
  • Proteins / analysis*
  • Proteins / chemistry
  • Proteins / genetics
  • Proteins / metabolism*
  • RNA, Transfer, Met / genetics
  • RNA, Transfer, Met / metabolism*
  • Staining and Labeling / methods*
  • Templates, Genetic


  • 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
  • Boron Compounds
  • Codon
  • Propionates
  • Proteins
  • RNA, Transfer, Met
  • propionic acid