ArticleProtein science : a publication of the Protein Society2013
Design, construction, and characterization of a second-generation DARP in library with reduced hydrophobicity.
Article in Protein science : a publication of the Protein Society, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 40 papers.
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40 citing papers in PubMed, 68 citations in OpenAlex.
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- Development of DARPin T cell engagers for specific targeting of tumor-associated HLA/peptide complexes.iScience · 2025Article
- Structural and Proteomic Analysis of the Mouse Cathepsin B-DARPin 4m3 Complex Reveals Species-Specific Binding Determinants.International journal of molecular sciences · 2025Article
- Optimal TELSAM-Target Protein Linker Character is Target Protein-Dependent.bioRxiv : the preprint server for biology · 2025Article
- Hyperstable Synthetic Mini-Proteins as Effective Ligand Scaffolds.ACS synthetic biology · 2023Article
- Fusion crystallization reveals the behavior of both the 1TEL crystallization chaperone and the TNK1 UBA domain.Structure (London, England : 1993) · 2023Article
- Chimeric antigen receptor-natural killer cells: a promising sword against insidious tumor cells.Human cell · 2023Review
- An Activation-Specific Anti-Mac-1 Designed-Ankyrin-Repeat-Protein Attenuates Colitis in Mice.Life (Basel, Switzerland) · 2023Article
- Fusion crystallization reveals the behavior of both the 1TEL crystallization chaperone and the TNK1 UBA domain.bioRxiv : the preprint server for biology · 2023Article
- Crystals of TELSAM-target protein fusions that exhibit minimal crystal contacts and lack direct inter-TELSAM contacts.Open biology · 2022Article
- Lentiviral and adeno-associated vectors efficiently transduce mouse T lymphocytes when targeted to murine CD8.Molecular therapy. Methods & clinical development · 2021Article
- Extending traditional antibody therapies: Novel discoveries in immunotherapy and clinical applications.Molecular therapy oncolytics · 2021Review
- High-throughput developability assays enable library-scale identification of producible protein scaffold variants.Proceedings of the National Academy of Sciences of the United States of America · 2021Article
- Driving CARs with alternative navigation tools - the potential of engineered binding scaffolds.The FEBS journal · 2021Review
- Mechanisms Applied by Protein Inhibitors to Inhibit Cysteine Proteases.International journal of molecular sciences · 2021Review
- The Effect of Mutations in the TPR and Ankyrin Families of Alpha Solenoid Repeat Proteins.Frontiers in bioinformatics · 2021Review
- Site-Specific, Stoichiometric-Controlled, PEGylated Conjugates of Fibroblast Growth Factor 2 (FGF2) with Hydrophilic Auristatin Y for Highly Selective Killing of Cancer Cells Overproducing Fibroblast Growth Factor Receptor 1 (FGFR1).Molecular pharmaceutics · 2020Article
- Generation of synthetic nanobodies against delicate proteins.Nature protocols · 2020Article
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Authors and funding
7 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Designed ankyrin repeat proteins (DARPins) are well-established binding molecules based on a highly stable nonantibody scaffold. Building on 13 crystal structures of DARPin-target complexes and stability measurements of DARPin mutants, we have generated a new DARPin library containing an extended randomized surface. To counteract the enrichment of unspecific hydrophobic binders during selections against difficult targets containing hydrophobic surfaces such as membrane proteins, the frequency of apolar residues at diversified positions was drastically reduced and substituted by an increased number of tyrosines. Ribosome display selections against two human caspases and membrane transporter AcrB yielded highly enriched pools of unique and strong DARPin binders which were mainly monomeric. We noted a prominent enrichment of tryptophan residues during binder selections. A crystal structure of a representative of this library in complex with caspase-7 visualizes the key roles of both tryptophans and tyrosines in providing target contacts. These aromatic and polar side chains thus substitute the apolar residues valine, leucine, isoleucine, methionine, and phenylalanine of the original DARPins. Our work describes biophysical and structural analyses required to extend existing binder scaffolds and simplifies an existing protocol for the assembly of highly diverse synthetic binder libraries.
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