PMID:16076451

Citation	Olejnik, J, Gite, S, Mamaev, S and Rothschild, KJ (2005) N-terminal labeling of proteins using initiator tRNA. Methods 36:252-60
Abstract	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).
Links	PubMed Online version:10.1016/j.ymeth.2005.04.003
Keywords	Escherichia coli/genetics; Fluorescent Dyes/chemistry; Protein Biosynthesis; Protein Engineering/methods; Proteins/chemistry; RNA, Transfer/chemistry; RNA, Transfer, Met/chemistry; Transfer RNA Aminoacylation
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