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Substitutional landscape of a split fluorescent protein fragment using high-density peptide microarrays

Antonescu, Oana N.; Rasmussen, Andreas; Damm, Nicole A. M.; Heidemann, Ditte F.; Popov, Roman; Nesterov-Mueller, Alexander; Johansson, Kristoffer E.; Winther, Jakob R.

Abstract:
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. ... mehr

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Verlagsausgabe §
DOI: 10.5445/IR/1000129508
Veröffentlicht am 09.02.2021
Originalveröffentlichung
DOI: 10.1371/journal.pone.0241461
Cover der Publikation
Zugehörige Institution(en) am KIT Institut für Mikrostrukturtechnik (IMT)
Publikationstyp Zeitschriftenaufsatz
Publikationsjahr 2021
Sprache Englisch
Identifikator ISSN: 1932-6203
KITopen-ID: 1000129508
Erschienen in PLOS ONE
Verlag Public Library of Science (PLoS)
Band 16
Heft 2
Seiten Article: e0241461
Vorab online veröffentlicht am 03.02.2021
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