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[1] Getting streptavidin to go it alone
DOI: 10.1038/nmeth861
Research in the April Nature Methods shows how to make a good thing even
better, modifying a popular system for protein labelling and modification to
reduce the risk of unwanted cross-reactions.
With incredible specificity and powerful affinity for each other, the
protein streptavidin and its small-molecule target biotin are truly the
'Dynamic Duo' of biological research, and a perennial favourite for use in
the design of biochemical experimental techniques. For example, one can
easily subject biotin-linked proteins to highly specific labelling with
streptavidin-linked fluorophores. Nonetheless, there is an important
limitation to the system-streptavidin naturally forms tetramers (assemblies
of four protein molecules) that bind up to four molecules of biotin,
creating the potential for unexpected cross-linking of biotinylated targets.
Efforts to engineer monomeric streptavidin variants have generally resulted
in diminished biotin affinity.
Alice Ting's lab now describes an alternative approach: engineering 'dead'
streptavidin variants that can bind to each other but not to biotin. By
combining the two types of streptavidin monomers in the proper proportions
and isolating tetramers that consist of three dead subunits and one active
subunit, they obtain streptavidin complexes that are functionally monomeric
and bind only one molecule of biotin. Experiments demonstrate that the
hybrid tetramers retain normal affinity for biotin but induce far less
'clumping' of biotinylated targets relative to wild-type streptavidin
tetramers.
This approach also offers the possibility of building divalent and trivalent
tetramers, and in an associated News & Views piece, Kai Johnsson comments
that "the existing plentitude of applications of the streptavidin-biotin
interaction provides an enormous playground for streptavidins with reduced
but defined valencies."
Author contact
Alice Y. Ting (Massachusetts Institute of Technology, Cambridge, MA, USA)
Tel: +1 617-452-2021; Email: [email protected]
Kai Johnsson (École Polytechnique Fédérale de Lausanne, Lausanne,
Switzerland)
Tel: +41 21 693 93 56; Email: [email protected]
[2] A new way to spotlight cells in live animals
DOI: 10.1038/nmeth868
Stanford University scientists have modified a popular strategy for imaging
studies in live animals, making possible a much broader range of biochemical
visualization experiments, as described in the April issue of Nature
Methods.
The processing of luciferin by the firefly enzyme luciferase generates
luminescence, and this luciferase-luciferin system has been used to monitor
a wide variety of cellular processes. Helen Blau and her colleagues now
introduce a new approach, sequential reporter-enzyme luminescence (SRL),
which uses Lugal, a non-toxic and cell-permeable luciferin variant that has
been chemically 'caged' so that luciferase cannot process it without the
prior action of the enzyme ?-galactosidase (?-gal). ?-gal is a robust enzyme
that can operate in a broad range of conditions, and it rapidly cleaves
Lugal to yield a compound that can act as a substrate for luciferase, making
luminescence dependent on the expression of both enzymes.
Blau's team show that by injecting Lugal into animals that express
luciferase throughout the body but express ?-gal from the promoter of a gene
with more tightly regulated expression, they can visually detect changes in
that gene's expression over time. With the large number of transgenic mice
available expressing ?-gal from different gene promoters, SRL offers a
promising tool for the real-time, in vivo study of the expression of many
genes. The authors also see the possibility that SRL could be adapted for
other detection platforms and conclude that "although the system described
here uses ?-gal, the concept of SRL should be readily applicable to other
commonly used enzymes."
Author contact
Helen M. Blau (Stanford University School of Medicine, Stanford, CA, USA)
Tel: +1 650-723-6209; Email: [email protected]
Other papers to be published in the March issue of Nature Methods:
[3] Imaging specific cell-surface proteolytic activity in single living cells
DOI: 10.1038/NMETH862
[4] Efficient incorporation of unnatural amino acids into proteins in Escherichia coli
DOI: 10.1038/NMETH864
[5] Slowed diffusion in tumors revealed by microfiberoptic epifluorescence photobleaching
DOI: 10.1038/NMETH863
[6] Genetically encoded fluorescent indicator for intracellular hydrogen peroxide
DOI: 10.1038/NMETH866
[7] Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8
DOI: 10.1038/NMETH865
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GEOGRAPHICAL LISTING OF AUTHORS
The following list of places refers to the whereabouts of authors on the
papers numbered in this release. The listing may be for an author's main
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the PDF of the paper for full details.
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SWITZERLAND
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San Francisco: 5
Stanford: 2
Illinois
Urbana: 1
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Bethesda: 3, 7
Massachusetts
Boston: 7
Cambridge: 1
Tennesse
Memphis: 7
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