- The twin-arginine translocation (TAT) pathway of the bacterial cytoplasmic membrane mediates translocation only of proteins that accomplished a native-like conformation. We deploy this feature in modular selection systems for directed evolution, in which folding helpers as well as dimeric or oligomeric proteinprotein interactions enable TAT-dependent translocation of the resistance marker TEM -lactamase (L). Specifically, we demonstrate and analyze selection of (i) enhancers for folding by direct TAT translocation selection of a target protein interposed between the TorA signal sequence and L, (ii) dimeric or oligomeric proteinprotein interactions by hitchhiker translocation (HiT) selection of proteins fused to the TorA signal sequence and to the L, respectively and (iii) heterotrimeric proteinprotein interactions by combining HiT with protein fragment complementation selection of proteins fused to two split L fragments and TorA, respectively. The lactamase fragments were additionally engineered for improved activity and stability.The twin-arginine translocation (TAT) pathway of the bacterial cytoplasmic membrane mediates translocation only of proteins that accomplished a native-like conformation. We deploy this feature in modular selection systems for directed evolution, in which folding helpers as well as dimeric or oligomeric proteinprotein interactions enable TAT-dependent translocation of the resistance marker TEM -lactamase (L). Specifically, we demonstrate and analyze selection of (i) enhancers for folding by direct TAT translocation selection of a target protein interposed between the TorA signal sequence and L, (ii) dimeric or oligomeric proteinprotein interactions by hitchhiker translocation (HiT) selection of proteins fused to the TorA signal sequence and to the L, respectively and (iii) heterotrimeric proteinprotein interactions by combining HiT with protein fragment complementation selection of proteins fused to two split L fragments and TorA, respectively. The lactamase fragments were additionally engineered for improved activity and stability. Applicability was benchmarked with interaction partners of known affinity and multimerization whereby cellular fitness correlated well with biophysical protein properties. Ultimately, the HiT selection was employed to identify peptides, which specifically bind to leukemia- and melanoma-relevant target proteins (MITF and ETO) by coiled-coil or tetra-helix-bundle formation with high affinity. The various versions of TAT selection led to inhibiting peptides (iPEPs) of disease-promoting interactions and enabled so far difficult to achieve selections.…
MetadatenVerfasserangaben: | Janina Speck, Christina Räuber, Tim Kükenshöner, Christoph Niemöller, Katelyn J. Mueller, Paula Schleberger, Padmarupa Dondapati, Jochen Hecky, Katja Maren ArndtORCiDGND, Kristian M. Müller |
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DOI: | https://doi.org/10.1093/protein/gzs098 |
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ISSN: | 1741-0126 |
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Titel des übergeordneten Werks (Englisch): | Protein engineering design & selection |
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Verlag: | Oxford Univ. Press |
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Verlagsort: | Oxford |
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Publikationstyp: | Wissenschaftlicher Artikel |
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Sprache: | Englisch |
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Jahr der Erstveröffentlichung: | 2013 |
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Erscheinungsjahr: | 2013 |
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Datum der Freischaltung: | 26.03.2017 |
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Freies Schlagwort / Tag: | HiT selection; NHR2; TAT selection; three hybrid; two hybrid |
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Band: | 26 |
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Ausgabe: | 3 |
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Seitenanzahl: | 18 |
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Erste Seite: | 225 |
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Letzte Seite: | 242 |
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Fördernde Institution: | Excellence Initiative of the German Federal and State Governments [EXC
294, BIOSS]; DFG [SPP1170]; Collaborative Research Centre (SFB) [850];
Deutsche Jose Carreras Leukamie-Stiftung [DJCLS R 07/10v, DJCLS R
06/12]; DAAD RISE Program |
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Organisationseinheiten: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie |
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Peer Review: | Referiert |
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