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. 2023 Jun 16;12(7):1935–1942. doi: 10.1021/acssynbio.3c00191

Figure 2.

Figure 2

NatA-mediated N-terminal acetylation of CPR1(10aa)(Q3E)-sfGFP. (A) SDS-PAGE analysis of CPR1(10aa)(Q3E)-sfGFP synthesized in the presence or absence of methionine aminopeptidase (MAP). (B) Schematic outline of NatA-mediated N-terminal acetylation. (C) SDS-PAGE analysis of CPR1(10aa)(Q3E)-sfGFP cosynthesized with yNaa10 and yNaa15. CPR1(10aa)(Q3E)-sfGFP DNA (5 nM), yNaa10 DNA (0.2 nM), and yNaa15 DNA (1 nM) were added to the PURE system ((−) 10-CHO-THF) containing 5 μM DnaK, 1 μM DnaJ, 1 μM GrpE, 1 μM MAP, and 1 mM acetyl-CoA. The reaction mixture was incubated at 23 °C for 24 h. (D) Extracted ion chromatograms of the methionine-excised N-terminal peptide derived from CPR1(10aa)(Q3E)-sfGFP after trypsin digestion (SEVYFDVEASK, M = 637.3010 Da, M+1 = 637.8025 Da, M+2 = 638.3038 Da, z = 2). The retention time was confirmed by the peptide search results from the MS/MS spectral search. (E) Extracted ion chromatograms of the methionine-excised and acetylated N-terminal peptide derived from CPR1(10aa)(Q3E)-sfGFP after trypsin digestion (S[Ace]-EVYFDVEASK, M = 658.3063 Da, M+1 = 658.8078 Da, M+2 = 659.3091 Da, z = 2). The retention time was confirmed by the peptide search results from the MS/MS spectral search.