Nature Methods 3, 267 - 273 (2006)
Published online: 22 March 2006; | doi:10.1038/nmeth861
A monovalent streptavidin with a single femtomolar biotin binding siteMark Howarth1, Daniel J-F Chinnapen1, 4, Kimberly Gerrow2, 4, Pieter C Dorrestein3, Melanie R Grandy1, Neil L Kelleher3, Alaa El-Husseini2
& Alice Y Ting11
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. 2
Department of Psychiatry, the Brain Research Center, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada. 3
Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, Illinois 61801, USA. 4
These authors contributed equally to this work.
Correspondence should be addressed to Alice Y Ting ating@mit.edu 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.
MORE ARTICLES LIKE THIS These links to content published by NPG are automatically generated.
|
