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A monovalent streptavidin with a single femtomolar biotin binding site


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.

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Figure 1: Generation of monovalent streptavidin.
Figure 2: Stability of monovalent streptavidin.
Figure 3: Affinity and off rate of biotin binding to chimeric tetramers.
Figure 4: Effect of monovalent and wild-type streptavidin on neuroligin-1 clustering.

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Funding was provided by the National Institutes of Health (1 R01 GM072670-01), the EJLB foundation, the Dreyfus foundation, the Sloan foundation and the Massachusetts Institute of Technology. M.H. was supported by a Computational and Systems Biology Initiative MIT–Merck postdoctoral fellowship, D.J.-F.C. by a National Science and Engineering Research Council of Canada postdoctoral fellowship, K.G. by a Michael Smith Foundation for Health Research student fellowship, P.C.D. by a National Institutes of Health Kirschstein NRSA postdoctoral fellowship, N.L.K. by the National Institutes of Health, and A.E.-H. by the Canadian Institutes for Health Research, the Michael Smith Foundation for Health Research, Neuroscience Canada and EJLB foundation. We thank P. Stayton for the streptavidin plasmid, Tanabe USA for biotin, J.A. Ryan for assistance with synthesis and T.S. Chen for preparing AP–neuroligin-1 and Ala–neuroligin-1 constructs.

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Correspondence to Alice Y Ting.

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Massachusetts Institute of Technology is seeking to file a patent application covering part of the information contained in this article.

Supplementary information

Supplementary Fig. 1

Mass spectrometry of chimeric streptavidins. (PDF 82 kb)

Supplementary Fig. 2

Labeling of site-specifically biotinylated cell surface proteins with monovalent streptavidin. (PDF 978 kb)

Supplementary Fig. 3

Additional examples of the effect of monovalent and wild-type streptavidin on neuroligin-1 clustering. (PDF 631 kb)

Supplementary Table 1

Neuroligin-1 clustering by streptavidin. (DOC 22 kb)

Supplementary Table 2

Effect of streptavidin on VGLUT1 clustering. (DOC 24 kb)

Supplementary Methods (DOC 72 kb)

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Howarth, M., Chinnapen, DF., Gerrow, K. et al. A monovalent streptavidin with a single femtomolar biotin binding site. Nat Methods 3, 267–273 (2006).

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