Fig. 1: A cell-free plate-based assay for detecting RRE-peptide interactions. | Nature Communications

Fig. 1: A cell-free plate-based assay for detecting RRE-peptide interactions.

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

Fig. 1

a Schematic of the cell-free workflow. sFLAG-tagged peptides and MBP-tagged RREs are expressed in individual PUREfrex reactions, mixed in a 384 well plate, and incubated to enable binding interactions. Addition of anti-FLAG AlphaLISA donor beads and anti-MBP AlphaLISA acceptor beads enables detection of interactions between the RRE and peptide of interest. PUREfrex reactions of precursor peptide and RRE for (b) pyrroloquinoline quinone (PQQ), (c) a putative lasso peptide from Thermobacculum terrenum ATCC BAA−798, (d) a heterocycloanthracin from Bacillus sp. Al Hakam, and (e) thiomuracin, a thiopeptide from Thermobispora bispora were cross-titrated across different dilutions (with 0 indicating no PUREfrex reaction added) and assessed for binding interactions via AlphaLISA. Data are representative of three (n = 3) biological replicates. RLU relative luminescence units. Source data are provided in the Source Data 1 file.

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