![]() Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M.Approaching infinite affinity through engineering of peptide-protein interaction. Keeble AH, Turkki P, Stokes S, Khairil Anuar INA, Rahikainen R, Hytönen VP, Howarth M.Extinction coefficient at 280 nm (predicted by ProtParam): 69,330 M -1 cm -1.To test the extent of disulfide bond formation, perform SDS-PAGE with Coomassie staining with and without DTT. This disulfide-bonded form will not be reactive with maleimide or other thiol-reactive reagents. As expected for exposed cysteines, Sp圜atcher003 (S49C) will eventually form a disulfide upon storage. Sp圜atcher003 (S49C): SDS-PAGE shows a 2nd band at slightly higher MW related to glucosylation, but this does not affect reactivity. Extinction coefficient at 280 nm (predicted by ProtParam): 17,420 M -1 cm -1.Sp圜atcher003: SDS-PAGE shows one higher band from gluconylation, which does not affect reactivity with SpyTag. Sp圜atcher003 reacts with SpyTag in the pH range 5-8 and is relatively insensitive to the buffer composition, as long as SDS or denaturants like urea or guanidinium are not used. Purified from Ni-NTA affinity chromatography and then by size exclusion chromatography.Ģ.32mg/mL (Sp圜atcher003), 1.8mg/mL (Sp圜atcher003, S49C), 2.47mg/mL (SpyTag003-MBP) SpyTag003: GSSHHHHHHSSGLVPRGSRGVPHIVMVDAYKRYKGSGESGKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSS Sp圜atcher003 (S49C): SYYHHHHHHDYDIPTTENLYFQGAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDCSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGSSGS ![]() Sp圜atcher003: SYYHHHHHHDYDIPTTENLYFQGAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGSSGS Sp圜atcher003 (S49C) has a unique cysteine for precise labeling with dye or precise attachment to surfaces or beads.įrom the laboratory of Mark Howarth, PhD, University of Oxford. SpyTag003 reacts with Sp圜atcher003 under a wide range of conditions and the after reaction the product is stable to boiling in SDS. SpyTag is a genetically encoded peptide (SpyTag003 sequence RGVPHIVMVDAYKRYK) that forms a spontaneous amide bond upon binding its genetically encoded partner Sp圜atcher (with the latest version Sp圜atcher003 available here). Peptide interaction with proteins is usually weak. Sp圜atcher (S49C) version contains a unique cysteine residue for precise labeling with dye or precise attachment to surfaces or beads.Extremely stable reaction product - Sp圜atcher/SpyTag complex is stable to boiling in SDS.Highly compatible - SpyTag reacts with Sp圜atcher under a wide range of conditions.SpyTag forms a spontaneous amide bond upon binding its genetically encoded partner Sp圜atcher.It is ideal for binding, labeling, immobilization and creating new kinds of protein architectures. The SpyTag/Sp圜atcher system is a convenient protein coupling tool for irreversible peptide-protein ligation.
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