Fig. 7: Site-specific dual incorporation of mTAF and pAcF into proteins for triple modification. | Nature Communications

Fig. 7: Site-specific dual incorporation of mTAF and pAcF into proteins for triple modification.

From: Noncanonical amino acids as doubly bio-orthogonal handles for one-pot preparation of protein multiconjugates

Fig. 7: Site-specific dual incorporation of mTAF and pAcF into proteins for triple modification.The alternative text for this image may have been generated using AI.

a Structure of mTAF. b SDS-PAGE analysis of site-specific incorporation of mTAF in response to UAG. Lanes 1 and 2 show mTAF-dependent production of sfGFP-Y151-mTAF-His×6. The purification experiments were repeated three times with reproducible results. c High-resolution mass spectrum of a purified sfGFP mutant containing mTAF mutant (Expected Mass 27743.96 Da, Observed Mass 27743.50 Da). d Schematic of strategy for encoding two ncAAs with two sets of orthogonal aaRS/tRNA pairs, which suppress the UAG and UAA stop codons to drive incorporation of pAcF and mTAF into proteins. e High-resolution mass spectrum of purified mutant scFv containing both mTAF and pAcF mutants (Expected Mass 27692.00 Da, Observed Mass 27692.00 Da). f SDS-PAGE of scFv-S9pAcF-K42mTAF after reaction with combinations of hydazide-CY5, DBCO-CY3, and TCO-PEG20K; images were obtained by Coomassie staining and in-gel fluorescence through chemoselective reactions. Assays were repeated three times with reproducible results. Source data of c and e are provided as a Source Data file.

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