| Autor: |
Kuo JC; Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, United States., Goudge MC; Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, New York 14853, United States., Metzloff AE; Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, United States., Huang LT; Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, United States., Colville MJ; Field of Biophysics, Cornell University, Ithaca, New York 14853, United States., Park S; Field of Biophysics, Cornell University, Ithaca, New York 14853, United States., Zipfel WR; Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, New York 14853, United States.; Field of Biophysics, Cornell University, Ithaca, New York 14853, United States.; Kavli Institute at Cornell for Nanoscale Science, Ithaca, New York 14853, United States., Paszek MJ; Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, United States.; Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, New York 14853, United States.; Field of Biophysics, Cornell University, Ithaca, New York 14853, United States.; Kavli Institute at Cornell for Nanoscale Science, Ithaca, New York 14853, United States. |
| Abstrakt: |
Precise pH measurements in the immediate environment of receptors is essential for elucidating the mechanisms through which local pH changes associated with diseased phenotypes manifest into aberrant receptor function. However, current pH sensors lack the ability to localize and target specific receptor molecules required to make these measurements. Herein we present the Litmus-body, our recombinant protein-based pH sensor, which through fusion to an anti-IgG nanobody is capable of piggybacking on IgG antibodies for molecular targeting to specific proteins on the cell surface. By normalizing a pH-dependent green fluorescent protein to a long Stokes shift red fluorophore or fluorescent protein, we readily report pH independent of sensor concentration using a single 488 nm excitation. Our Litmus-body showed excellent responsiveness in solution, with a greater than 50-fold change across the regime of physiological pH. The sensor was further validated for use on live cells and shown to be specific to the protein of interest. In complex with our Litmus-body, cetuximab therapeutic antibody retained its functionality in binding and inhibiting ligand interaction of its target epidermal growth factor receptor (EGFR), triggering receptor-mediated endocytosis that allowed tracking of local pH from the cell surface through the endocytic pathway. |
