Designing Repeat Proteins: Well-expressed, Soluble and Stable Proteins from Combinatorial Libraries of Consensus Ankyrin Repeat Proteins
Abstract
We describe an efficient way to generate combinatorial libraries of stable, soluble and well-expressed ankyrin repeat (AR) proteins. Using a combination of sequence and structure consensus analyses, we designed a 33 amino acid residue AR module with seven randomized positions having a theoretical diversity of 7.2x10(7). Different numbers of this module were cloned between N and C-terminal capping repeats, i.e. ARs designed to shield the hydrophobic core of stacked AR modules. In this manner, combinatorial libraries of designed AR proteins consisting of four to six repeats were generated, thereby potentiating the theoretical diversity. All randomly chosen library members were expressed in soluble form in the cytoplasm of Escherichia coli in amounts up to 200 mg per 1 l of shake-flask culture. Virtually pure proteins were obtained in a single purification step. The designed AR proteins are monomeric and display CD spectra identical with those of natural AR proteins. At the same time, our AR proteins are highly thermostable, with T(m) values ranging from 66 degrees C to well above 85 degrees C. Thus, our combinatorial library members possess the properties required for biotechnological applications. Moreover, the favorable biophysical properties and the modularity of the AR fold may account, partly, for the abundance of natural AR proteins.
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POI-targeting polypeptides can either be single chain antibodies (e.g. nanobodies) or a variety of synthetic polypeptide scaffolds that bind POIs such as monobodies, DARPINs, affimers, or Affibodies.
“These POI-targeting polypeptides can either be single chain antibodies (e.g. variable domains from camelid or shark-derived heavy chain only antibodies, nanobodies), or a variety of synthetic polypeptide scaffolds that bind POIs such as fibronectin type III domain-based monobodies [110, 111], designed ankyrin repeats (DARPINs) [112, 113], cystatin-derived affimers [114] or Affibodies derived from”
From Advances in targeted degradation of endogenous proteins (2019) · cites this paper as [112]
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- Authors
- Binz, H. K., Stumpp, M. T., Forrer, P., Amstutz, P., & Plückthun, A.
- Affiliations
- University of Zurich (CH)
- Corresponding author
- Plückthun, Andreas
- Venue
- Journal of Molecular Biology
- Publisher
- Elsevier BV
- Published
- 2003-09
- Volume
- 332
- Issue
- 2
- Pages
- 489-503
- ISSN
- 0022-2836 (ISSN-L)
- OpenAlex
- W2092803887
- Cited by
- 589
- References
- 57
- Citations by year
- 2026: 16 · 2025: 22 · 2024: 13 · 2023: 29 · 2022: 24 · 2021: 28 · 2020: 27 · 2019: 32 · 2018: 26 · 2017: 40 · 2016: 22 · 2015: 28 · 2014: 37 · 2013: 36 · 2012: 28
- FWCI
- 5.75
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Molecular Biology (publishedVersion)
- PubMed (publishedVersion)
- Funders
- MorphoSys
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chemical Synthesis and Analysis
- Keywords
- ankyrin repeat proteins, protein design, combinatorial libraries, protein stability, thermal stability, protein solubility, Escherichia coli expression, protein folding, monomeric protein
- MeSH terms
- Amino Acid Sequence, Base Sequence, Circular Dichroism, Models, Molecular, Molecular Sequence Data, Temperature, Protein Engineering, Gene Library, Consensus Sequence, Ankyrin Repeat, Protein Structure, Tertiary, Repetitive Sequences, Amino Acid, Databases, Protein
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