Split fluorescent proteins have wide applicability as biosensors for protein-protein interactions, genetically encoded tags for protein detection and localization, as well as fusion partners in super-resolution microscopy. We have here established and validated a novel platform for functional analysis of leave-one-out split fluorescent proteins (LOO-FPs) in high throughput and with rapid turnover. We have screened more than 12,000 variants of the beta-strand split fragment using high-density peptide microarrays for binding and functional complementation in Green Fluorescent Protein. We studied the effect of peptide length and the effect of different linkers to the solid support. We further mapped the effect of all possible amino acid substitutions on each position as well as in the context of some single and double amino acid substitutions. As all peptides were tested in 12 duplicates, the analysis rests on a firm statistical basis allowing for confirmation of the robustness and precision of the method. Based on experiments in solution, we conclude that under the given conditions, the signal intensity on the peptide microarray faithfully reflects the binding affinity between the split fragments. With this, we are able to identify a peptide with 9-fold higher affinity than the starting peptide.
Split fluorescent proteins have wide applicability as biosensors for protein-protein interactions, genetically encoded tags for protein detection and localization, as well as fusion partners in superresolution microscopy. In this work, we have established and validated a novel platform for functional analysis of leave-one-out split fluorescent proteins (LOO-FPs) in high throughput and with rapid turnover. We have screened more than 12,000 complementing peptide variants using high-density peptide microarrays and examined them for binding and functional complementation of the LOO-FP fragment into Green Fluorescent Protein. We have studied the effect of peptide length and the effect of different linkers to the solid support. The peptide microarray platform allowed us to map the effect of all possible amino acid substitutions on each position of the complementing peptides as well as in the context of some single and double amino acid substitutions. As all peptides in different formats were tested in 12 duplicates, the analysis rests on a firm statistical basis allowing determination of robustness and precision of the method.Importantly, we showed that the microarray fluorescence correlated with the affinity in solution between the LOO-FP and peptides. A double substitution yielded a peptide with 9-fold higher affinity than the starting peptide.
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