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. Author manuscript; available in PMC: 2023 Sep 1.
Published in final edited form as: Biotechnol J. 2022 Jun 9;17(9):e2200096. doi: 10.1002/biot.202200096

FIGURE 5.

FIGURE 5

Top-down mass spectrometry demonstrates efficient incorporation of ncAAs into sfGFP. pLysN and ProCarb were incorporated into sfGFP using the indicated extracts. sfGFP variants containing (A) 1TAG (sfGFPT216), (B) 2TAG (sfGFP S2T216) or 3TAG (sfGFPS2N212T216) were synthesized by the various extracts as indicated. In each example, colored peaks correspond to the expected mass. The second peak under the red arrow in panel (A) corresponds to the mass of the modified protein retaining the initiator methionine residue. All other samples have this methionine cleaved off. Exact mass and mass shifts are indicated in Supplementary Table S1. Data representative of three independent reactions. ncAAs, noncanonical amino acids; sfGFP, superfolder green fluorescent protein