Bifunctional avidin with covalently modifiable ligand binding site

Jenni Leppiniemi, Juha A.E. Määttä, Henrik Hammaren, Mikko Soikkeli, Mikko Laitaoja, Janne Jänis, Markku S. Kulomaa, Vesa P. Hytönen

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13 Citations (Scopus)


The extensive use of avidin and streptavidin in life sciences originates from the extraordinary tight biotin-binding affinity of these tetrameric proteins. Numerous studies have been performed to modify the biotin-binding affinity of (strept)avidin to improve the existing applications. Even so, (strept)avidin greatly favours its natural ligand, biotin. Here we engineered the biotin-binding pocket of avidin with a single point mutation S16C and thus introduced a chemically active thiol group, which could be covalently coupled with thiol-reactive molecules. This approach was applied to the previously reported bivalent dual chain avidin by modifying one binding site while preserving the other one intact. Maleimide was then coupled to the modified binding site resulting in a decrease in biotin affinity. Furthermore, we showed that this thiol could be covalently coupled to other maleimide derivatives, for instance fluorescent labels, allowing intratetrameric FRET. The bifunctional avidins described here provide improved and novel tools for applications such as the biofunctionalization of surfaces.

Original languageEnglish
Article numbere16576
Pages (from-to)e16576
JournalPLoS One
Issue number1
Publication statusPublished - 2011
Publication typeA1 Journal article-refereed

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