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Our general method for the peptide quantification was as follows: Radioisotope (RI) counts of [14C]-Asp were plotted against its known concentrations to determine the calibration line. Then, RI counts of the expressed wild-type peptide and cyclic peptides were fitted to the calibration line to estimate the individual concentration of peptides. We found that this quantification method was reliable to assess the quantity of peptide previously proven by performing activity assays using a known bioactive peptide. 6
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During the translation, the azide group of Aha, Anv, and Anl was very likely reduced by DTT and/or mercaptoethanol included in the translation buffer. See the following reference as an example of chemistry: Meinjohanns, E.; Meldal, M.; Jensen, T.; Werdelin, O.; GalliStampino, L.; Mouritsen, S.; Bock, K. J. Chem. Soc., Perkin Trans. 1 1997, 871-884.
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During the translation, the azide group of Aha, Anv, and Anl was very likely reduced by DTT and/or mercaptoethanol included in the translation buffer. See the following reference as an example of chemistry: Meinjohanns, E.; Meldal, M.; Jensen, T.; Werdelin, O.; GalliStampino, L.; Mouritsen, S.; Bock, K. J. Chem. Soc., Perkin Trans. 1 1997, 871-884.
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Back, J. W.; David, O.; Kramer, G.; Masson, G.; Kasper, P. T.; de Koning, L. J.; de Jong, L.; van Maarseveen, J. H.; de Koster, C. G. Angew. Chem., Int. Ed. 2005, 44, 7946-7950. We found that the reported mechanism herein also proceeds in the translated peptides containing appropriate nonproteinogenic amino acids (Nakajima, E.; et al. unpublished results). More detailed studies of this work will be reported elsewhere.
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Back, J. W.; David, O.; Kramer, G.; Masson, G.; Kasper, P. T.; de Koning, L. J.; de Jong, L.; van Maarseveen, J. H.; de Koster, C. G. Angew. Chem., Int. Ed. 2005, 44, 7946-7950. We found that the reported mechanism herein also proceeds in the translated peptides containing appropriate nonproteinogenic amino acids (Nakajima, E.; et al. unpublished results). More detailed studies of this work will be reported elsewhere.
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