TY  - JOUR
A1  - Speck, Janina
A1  - Räuber, Christina
A1  - Kükenshöner, Tim
A1  - Niemöller, Christoph
A1  - Mueller, Katelyn J.
A1  - Schleberger, Paula
A1  - Dondapati, Padmarupa
A1  - Hecky, Jochen
A1  - Arndt, Katja Maren
A1  - Müller, Kristian M.
T1  - TAT hitchhiker selection expanded to folding helpers, multimeric interactions and combinations with protein fragment complementation
JF  - Protein engineering design & selection
N2  - 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.
KW  - HiT selection
KW  - NHR2
KW  - TAT selection
KW  - three hybrid
KW  - two hybrid
Y1  - 2013
U6  - https://doi.org/10.1093/protein/gzs098
SN  - 1741-0126
VL  - 26
IS  - 3
SP  - 225
EP  - 242
PB  - Oxford Univ. Press
CY  - Oxford
ER  -