FIGURE 2.

Preparation of fluorescently labeled KaiB. a, a schematic representation of TAMRA-KaiB-His₆ protein synthesis using position-specific incorporation of a fluorescent amino acid in a cell-free translation system with an expanded genetic code. TAMRA-KaiB-His₆ is designed to have 16 amino acid residues at the N terminus, including a TAMRA-X-aminophenylalanine residue at the ninth amino acid position, 102 amino acid KaiB residues, and a histidine tag in the 20 amino acid residues at the C terminus (19). b, detecting TAMRA-KaiB-His₆ using SDS-PAGE. A 2-μl aliquot of each fraction after gel-filtration purification was examined on a 10–20% gradient gel. TAMRA in the gel was imaged using a Typhoon 9400 system (GE Healthcare). Approximate protein sizes are shown. c, KaiC phosphorylation in the oscillatory solution containing fluorescently labeled KaiB. The standard mixture was prepared with or without TAMRA-KaiB-His₆ and incubated at 30 °C (5). Aliquots were collected every 4 h and subjected to SDS-PAGE analysis to measure KaiC phosphorylation levels. Filled and open circles indicate the ratios of phosphorylated KaiC to total KaiC in the standard mixture with and without TAMRA-KaiB-His₆, respectively.