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- Title
Modification of the loops in the ligand-binding site turns avidin into a steroid-binding protein.
- Authors
Riihimäki, Tiina A.; Hiltunen, Soili; Rangl, Martina; Nordlund, Henri R.; Määttä, Juha A. E.; Ebner, Andreas; Hinterdorfer, Peter; Kulomaa, Markku S.; Takkinen, Kristiina; Hytönen, Vesa P.
- Abstract
Background: Engineered proteins, with non-immunoglobulin scaffolds, have become an important alternative to antibodies in many biotechnical and therapeutic applications. When compared to antibodies, tailored proteins may provide advantageous properties such as a smaller size or a more stable structure. Results: Avidin is a widely used protein in biomedicine and biotechnology. To tailor the binding properties of avidin, we have designed a sequence-randomized avidin library with mutagenesis focused at the loop area of the binding site. Selection from the generated library led to the isolation of a steroid-binding avidin mutant (sbAvd-1) showing micromolar affinity towards testosterone (Kd ∼ 9 μM). Furthermore, a gene library based on the sbAvd-1 gene was created by randomizing the loop area between β-strands 3 and 4. Phage display selection from this library led to the isolation of a steroid-binding protein with significantly decreased biotin binding affinity compared to sbAvd-1. Importantly, differential scanning calorimetry and analytical gel-filtration revealed that the high stability and the tetrameric structure were preserved in these engineered avidins. Conclusions: The high stability and structural properties of avidin make it an attractive molecule for the engineering of novel receptors. This methodology may allow the use of avidin as a universal scaffold in the development of novel receptors for small molecules.
- Subjects
BIOTIN; AVIDIN; STEROID-binding proteins; IMMUNOGLOBULINS; BIOMOLECULES
- Publication
BMC Biotechnology, 2011, Vol 11, Issue 1, p64
- ISSN
1472-6750
- Publication type
Article
- DOI
10.1186/1472-6750-11-64