Incorporation of Non-Natural Amino Acids with Two Labeling Groups into the N-Terminus of Proteins

Incorporation of non-natural amino acids into proteins is a useful method for protein researches, including position-specific labeling with fluorophores and biotin. Here, we incorporated single non-natural amino acids with both fluorophore and biotin into the N-terminus of proteins in a cell-free translation system. alpha-Biotinyl-p-(BODIPYFL-amino)phenylalanine derivatives, with or without an aminohexyl linker between the biotin moiety, were synthesized and attached to an Escherichia coli initiator tRNA that had a CUA anticodon. The aminoacylated initiator tRNA was added to an E. coli cell-free translation system together with an mRNA encoding maltose binding protein that had a UAG initiator codon. Fluorescence analysis of SDS-PAGE, Western blot analysis, and mass spectrometry demonstrated that the biotinylated BODIPYFL-aminophenylalanine derivatives were successfully incorporated into the N-terminus of the protein, although the aminohexyl linker slightly decreased the incorporation efficiency. Fluorescence spectroscopy measurements indicated that complexation with streptavidin significantly quenched the fluorescence of BODIPYFL, and the aminohexyl linker facilitated the fluorescence quenching. The biotinylated BODIPYFL-aminophenylalanine was also used for double labeling several proteins. This method is a general and useful tool for the N-terminal-specific modification of proteins with two moieties.