Proceedings book download - 5Z.com

Fig. 1. Photoaffinity labeling. Fig. 2A. CNBr digestion.Fig. 2B. Enzymatic digestion.to Glu C enzymatic digestion that should highlight larger migrated fragments (seeFigure 2B). Again this digestion showed an elevation of 5 to 6 kDa on SDS-PAGEelectrophoresis. We can also exclude intramolecular cross-linking due to the fact that onlythe intracellular fragment of TMD 4 could account for the CNBr digestion’s gain in mass,yet according to molecular models, this region is over 20 Å away from the Bpa and thus toofar to form a covalent bond even at extreme temperatures [5].We have shown that the expression of hAT 1 containing the non-canonical amino acidBpa can be accomplished with reasonable yields. These receptors can easily be identifiedusing photoaffinity labeling. We are also able to identify an auxiliary hAT 1 binding protein,which has at least one CNBr digestion fragment of 1 to 2 kDa and a Glu C digestionfragment of 5 to 6 kDa. Subsequent immunoblotting experiments could be used topositively identify this hAT 1 binding protein. Furthermore, this method can easily beextrapolated to use other non-canonical amino acids that have been developed by the P.G.Schultz group by using the same tRNA and mutated receptor sequences.AcknowledgmentsWe thank Marie-Reine Lefebvre for her expert knowledge and expertise. Funding was supported bythe CIHR.References1. Liu, W., et al. Nature Methods 4(3), 239-244 (2007).2. Perodin, J., et al. Biochemistry 41(48), 14348-14356 (2002).3. Rihakova, L., et al. J. Recept Signal Transduct. Res. 22(1-4), 297-313 (2002).4. Fillion, D., et al. J. Med. Chem. 55(5), 2073-2075 (2010).5. Arsenault, J., et al. Biochem. Pharmacol. 80(7), 990-999 (2010).547