@article{SpeckRaeuberKuekenshoeneretal.2013,
  author    = {Speck, Janina and R{\"a}uber, Christina and K{\"u}kensh{\"o}ner, Tim and Niem{\"o}ller, Christoph and Mueller, Katelyn J. and Schleberger, Paula and Dondapati, Padmarupa and Hecky, Jochen and Arndt, Katja Maren and M{\"u}ller, Kristian M.},
  title     = {TAT hitchhiker selection expanded to folding helpers, multimeric interactions and combinations with protein fragment complementation},
  series = {Protein engineering design \& selection},
  volume    = {26},
  journal   = {Protein engineering design \& selection},
  number    = {3},
  publisher = {Oxford Univ. Press},
  address   = {Oxford},
  issn      = {1741-0126},
  doi       = {10.1093/protein/gzs098},
  pages     = {225 -- 242},
  year      = {2013},
  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 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.},
  language  = {en}
}
@article{KuekenshoenerWohlwendNiemoelleretal.2014,
  author    = {Kuekenshoener, Tim and Wohlwend, Daniel and Niemoeller, Christoph and Dondapati, Padmarupa and Speck, Janina and Adeniran, Adebola V. and Nieth, Anita and Gerhardt, Stefan and Einsle, Oliver and Mueller, Kristian M. and Arndt, Katja Maren},
  title     = {Improving coiled coil stability while maintaining specificity by a bacterial hitchhiker selection system},
  series = {Journal of structural biology},
  volume    = {186},
  journal   = {Journal of structural biology},
  number    = {3},
  publisher = {Elsevier},
  address   = {San Diego},
  issn      = {1047-8477},
  doi       = {10.1016/j.jsb.2014.03.002},
  pages     = {335 -- 348},
  year      = {2014},
  abstract  = {The design and selection of peptides targeting cellular proteins is challenging and often yields candidates with undesired properties. Therefore we deployed a new selection system based on the twin-arginine translocase (TAT) pathway of Escherichia coli, named hitchhiker translocation (HiT) selection. A pool of alpha-helix encoding sequences was designed and selected for interference with the coiled coil domain (CC) of a melanoma-associated basic-helix-loop-helix-leucine-zipper (bHLHLZ) protein, the microphthalmia associated transcription factor (MITF). One predominant sequence (iM10) was enriched during selection and showed remarkable protease resistance, high solubility and thermal stability while maintaining its specificity. Furthermore, it exhibited nanomolar range affinity towards the target peptide. A mutation screen indicated that target-binding helices of increased homodimer stability and improved expression rates were preferred in the selection process. The crystal structure of the iM10/MITF-CC heterodimer (2.1 angstrom) provided important structural insights and validated our design predictions. Importantly, iM10 did not only bind to the MITF coiled coil, but also to the markedly more stable HLHLZ domain of MITF. Characterizing the selected variants of the semi-rational library demonstrated the potential of the innovative bacterial selection approach. (C) 2014 Elsevier Inc. All rights reserved.},
  language  = {en}
}