Figure 6. Position-specific incorporation of fluorescent amino acids by the cell-free system.

RNA templates encoding variants of KvAP, pro-TNF, or Syb2 were translated in the cell-free system supplemented with BODIPYFL-AF conjugated tRNA with or without the addition of Sf21microsomes (microsomes). The non-natural amino acids were incorporated into introduced TAG codon. The resulting proteins were subjected to SDS-PAGE (10-15% polyacrylamide) and detected with excitation at 488 nm and emission at 530 nm. The amber codon in the different constructs was substituted for the codon at the position corresponding to the listed amino acids (except for M6 in pro-TNF where the TAG codon was inserted as an additional codon after the M6 codon) and marked by a star in the diagram.