title: TAT hitchhiker selection expanded to folding helpers, multimeric interactions and combinations with protein fragment complementation.
abstract: 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 protein-protein interactions enable TAT-dependent translocation of the resistance marker TEM β-lactamase (βL). 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 βL, (ii) dimeric or oligomeric protein-protein interactions by hitchhiker translocation (HiT) selection of proteins fused to the TorA signal sequence and to the βL, respectively and (iii) heterotrimeric protein-protein interactions by combining HiT with protein fragment complementation selection of proteins fused to two split βL 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.
authors.affiliation: Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany.
authors.forename: Janina
authors.initials: J
authors.lastname: Speck
authors.forename: Christina
authors.initials: C
authors.lastname: Räuber
authors.forename: Tim
authors.initials: T
authors.lastname: Kükenshöner
authors.forename: Christoph
authors.initials: C
authors.lastname: Niemöller
authors.forename: Katelyn J
authors.initials: KJ
authors.lastname: Mueller
authors.forename: Paula
authors.initials: P
authors.lastname: Schleberger
authors.forename: Padmarupa
authors.initials: P
authors.lastname: Dondapati
authors.forename: Jochen
authors.initials: J
authors.lastname: Hecky
authors.forename: Katja M
authors.initials: KM
authors.lastname: Arndt
authors.forename: Kristian M
authors.initials: KM
authors.lastname: Müller
mesh_terms: D001120:Arginine / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D001120:Arginine; D001618:beta-Lactamases