Nature Methods

Research in the April Nature Methods shows how to make a good thing even better, modifying a popular system for protein labeling 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 favorite for use in the design of biochemical experimental techniques. For example, one can easily subject biotin-linked proteins to highly specific labeling 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."

CONTACT
Alice Y. Ting (Massachusetts Institute of Technology, Cambridge, MA, USA)
Tel: +1 617 452 2021; Email: ating@mit.edu


Kai Johnsson (École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland)
Tel: +41 21 693 93 56; Email: kai.johnsson@epfl.ch




A new way to spotlight cells in live animals

Nature Methods

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 β-galactosidase (β-gal). β-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 β-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 &beta-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 ß-gal, the concept of SRL should be readily applicable to other commonly used enzymes."

CONTACT
Helen M. Blau (University School of Medicine, Stanford, CA, USA)
Tel: +1 650 723 6209; Email: hblau@stanford.edu

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