Incorporation of Fluorescent-Labeled Non-α-Amino Carboxylic Acids into the N-Terminus of Proteins in Response to Amber Initiation Codon

Abstract

Abstract Incorporation of non-natural amino acid derivatives containing fluorescent groups into proteins is a useful method for protein analyses. Here, we investigated the incorporation of fluorescent-labeled non-α-amino carboxylic acids into the N-terminus of proteins in response to the UAG initiation codon. A series of TAMRA-labeled amino carboxylic acids were synthesized and attached to an amber suppressor initiator tRNA derived from Escherichia coli initiator tRNA. Fluorescent-labeled amino carboxylic acids were successfully incorporated into the N-terminus of streptavidin by adding TAMRA-acylated initiator tRNAs to an E. coli cell-free translation system, although the incorporation efficiency differed depending on the amino carboxylic acid chain length. Based on this observation, 3-aminopropionic acid derivatives labeled with BODIPY, rhodamine, and cyanine fluorophores were designed and synthesized. The fluorescent-labeled 3-aminopropionic acid derivatives developed using BODIPY and rhodamine dyes could be incorporated with good efficiency. On the other hand, 6-aminohexanoic acid was effectively incorporated when labeled with cyanine dyes. The present study demonstrates that translation initiation can accept a wide variety of non-natural substrates and provides a useful method for N-terminal-specific labeling of proteins with various fluorophores.