SLAS

MP152:Cosentino:AlphaLISA StrepTactin Protein-Protein Interactions

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Development of Strep-Tactin-conjugated Alpha Donor and AlphaLISA Acceptor beads as new tools for homogenous custom-based protein-protein interaction assays


Gregory Cosentino, Nancy Mac Donald, Marie Boulé, Marie-Hélène Venne, Thomas Lassalle, Anja Rodenbrock, Stéphane Parent, Véronique Brechler and Lenka Rihakova
Bio-discovery, PerkinElmer, 1744 William, Suite 600, Montréal, Québec, Canada H3J 1R4

Alpha and AlphaLISA® Toolbox products are stand-alone bead reagents developed to enable custom building of assays. These reagents have been used to study a wide variety of biochemical and cellular events including protein-protein interactions. The Toolbox is comprised of Alpha Donor and AlphaLISA Acceptor beads coated with different molecules depending on the nature of the assay under study. In a protein-protein interaction assay, one protein is captured on the Donor beads and the other protein is captured on the Acceptor beads. When the two proteins interact, the Donor bead is brought into proximity of the Acceptor bead, and subsequent excitation of the Donor bead will result in chemical energy transfer to the Acceptor bead generating a luminescent signal.


This work presents the development of Strep-Tactin®-conjugated Alpha Donor and AlphaLISA Acceptor beads. The Strep-Tactin®/Strep-tag® system is a unique protein purification and detection system based on high selectivity of the engineered streptavidin mutant Strep-Tactin® for Strep-tag® peptide tags, which can be introduced within the sequence of any recombinant protein. Strep-Tactin® was conjugated to Alpha Donor beads or AlphaLISA Acceptor beads and tested in an AlphaLISA assay using double tagged protein probe (Strep-tag II-Azurin-6xHis and One-STrEP tag-Azurin-6xHis). Following optimization of the assay parameters (e.g. order of addition of the assay reagents), results show that Strep-Tactin®-conjugated beads recognize Strep tag II-tagged proteins with nearly 100-fold higher apparent affinity compared to streptavidin-conjugated equivalents, while the apparent affinity for biotin-labeled proteins is the same for both streptavidin and Strep-Tactin®-conjugated beads. In addition, Alpha Donor beads and AlphaLISA Acceptor beads conjugated to Strep-Tactin® can be used in the presence of cell lysates or some cell culture media without impacting assay performance parameters, such as EC50 and signal-to-noise ratio. Overall, Strep-Tactin®-conjugated beads, together with other new Toolbox products (pre-coated beads able to capture common protein affinity tags such as FLAG®, MBP, 6xHis, or FITC, as well as beads conjugated with several anti-species antibodies) are versatile tools for custom-based homogenous interaction assays.

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