A monovalent streptavidin with a single femtomolar biotin binding site

Mark Howarth, Daniel J F Chinnapen, Kimberly Gerrow, Pieter C. Dorrestein, Melanie R. Grandy, Neil L. Kelleher, Alaa El-Husseini, Alice Y. Ting*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

245 Scopus citations

Abstract

Streptavidin and avidin are used ubiquitously because of the remarkable affinity of their biotin binding, but they are tetramers, which disrupts many of their applications. Making either protein monomeric reduces affinity by at least 104-fold because part of the binding site comes from a neighboring subunit. Here we engineered a streptavidin tetramer with only one functional biotin binding subunit that retained the affinity, off rate and thermostability of wild-type streptavidin. In denaturant, we mixed a streptavidin variant containing three mutations that block biotin binding with wild-type streptavidin in a 3:1 ratio. Then we generated monovalent streptavidin by refolding and nickel-affinity purification. Similarly, we purified defined tetramers with two or three biotin binding subunits. Labeling of site-specifically biotinylated neuroligin-1 with monovalent streptavidin allowed stable neuroligin-1 tracking without cross-linking, whereas wild-type streptavidin aggregated neuroligin-1 and disrupted presynaptic contacts. Monovalent streptavidin should find general application in biomolecule labeling, single-particle tracking and nanotechnology.

Original languageEnglish (US)
Pages (from-to)267-273
Number of pages7
JournalNature Methods
Volume3
Issue number4
DOIs
StatePublished - Apr 2006

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'A monovalent streptavidin with a single femtomolar biotin binding site'. Together they form a unique fingerprint.

Cite this