Fig. 2: Cell-free workflow identifies peptide residues important for binding by TbtF. | Nature Communications

Fig. 2: Cell-free workflow identifies peptide residues important for binding by TbtF.

From: Characterizing and engineering post-translational modifications with high-throughput cell-free expression

Fig. 2

a An alanine scan library of the leader sequence of TbtA was expressed in individual PUREfrex reactions and assessed for binding interactions in the presence of MBP-TbtF RRE domain using AlphaLISA. b A synthetic peptide library was constructed using the first 40 amino acids of sfGFP. Variants of the sfGFP were then constructed by replacing residues in the peptide identified in the alanine scan as important for binding by TbtF with the corresponding residue in the wild-type TbtA leader sequence. Each peptide variant was expressed in an individual PUREfrex reaction and then assessed for binding interactions in the presence and absence of TbtF using AlphaLISA. Peptide variant 2 contains 9% identity to TbtA wild-type peptide due to sharing residues G(-2), G(-7) and G(−9). For simplicity, only amino acids between the −34 and −17 position are depicted, however each peptide was composed of 40 amino acids reflecting the length of the TbtA leader sequence with an additional 5 amino acid linker. Sequences for each of the peptide variants assayed in panel b are provided in Supplementary Table 1. All data are presented as the mean of technical replicates (n = 3). RLU relative luminescence units. Source data are provided in the Source Data 1 file.

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