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BMC Biotechnol. 2011 Jun 9;11:64. doi: 10.1186/1472-6750-11-64.

Modification of the loops in the ligand-binding site turns avidin into a steroid-binding protein.

Author information

  • 1Institute of Biomedical Technology, University of Tampere and Tampere University Hospital, FI-33520 Tampere, Finland.

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.

PMID:
21658230
[PubMed - indexed for MEDLINE]
PMCID:
PMC3201017
Free PMC Article

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