Real-time monitoring of vesicle pH in an endocytic pathway using an EGF-conjugated two-photon probe.

Herein, we developed a ratiometric two-photon probe (BHS3-EGF), derived from a pH sensitive dye and epidermal growth factor (EGF), for real-time monitoring and quantitative analysis of acidic luminal pH values during endocytic pathway activity. Two-photon microscopy imaging with BHS3-EGF allows the quantitative analysis of pH distributions of single vesicles and their dynamics in receptor-mediated endocytosis in real-time.

Incorporation of Unnatural Amino Acids in Response to the AGG Codon.

The biological protein synthesis system has been engineered to incorporate unnatural amino acid into proteins, and this has opened up new routes for engineering proteins with novel compositions. While such systems have been successfully applied in research, there remains a need to develop new approaches with respect to the wider application of unnatural amino acids. In this study, we reported a strategy for incorporating unnatural amino acids into proteins by reassigning one of the Arg sense codons, the AGG codon. Using this method, several unnatural amino acids were quantitatively incorporated into the AGG site. Furthermore, we applied the method to multiple AGG sites, and even to tandem AGG sequences. The method developed and described here could be used for engineering proteins with diverse unnatural amino acids, particularly when employed in combination with other methods.