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Open AccessArticle

A Recombinant Affinity Reagent Specific for a Phosphoepitope of Akt1

Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607, USA
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Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2018, 19(11), 3305; https://doi.org/10.3390/ijms19113305
Received: 17 August 2018 / Revised: 10 October 2018 / Accepted: 16 October 2018 / Published: 24 October 2018
(This article belongs to the Special Issue Recombinant Proteins)
The serine/threonine-protein kinase, Akt1, plays an important part in mammalian cell growth, proliferation, migration and angiogenesis, and becomes activated through phosphorylation. To monitor phosphorylation of threonine 308 in Akt1, we developed a recombinant phosphothreonine-binding domain (pTBD) that is highly selective for the Akt1 phosphopeptide. A phage-display library of variants of the Forkhead-associated 1 (FHA1) domain of yeast Rad53p was screened by affinity selection to the phosphopeptide, 301-KDGATMKpTFCGTPEY-315, and yielded 12 binding clones. The strongest binders have equilibrium dissociation constants of 160–180 nanomolar and are phosphothreonine-specific in binding. The specificity of one Akt1-pTBD was compared to commercially available polyclonal antibodies (pAbs) generated against the same phosphopeptide. The Akt1-pTBD was either equal to or better than three pAbs in detecting the Akt1 pT308 phosphopeptide in ELISAs. View Full-Text
Keywords: affinity selection; alanine-scanning; Forkhead Associated (FHA) domain; phosphopeptide; phage-display; surface plasmon resonance (SPR) affinity selection; alanine-scanning; Forkhead Associated (FHA) domain; phosphopeptide; phage-display; surface plasmon resonance (SPR)
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McGinnis, J.E.; Venegas, L.A.; Lopez, H.; Kay, B.K. A Recombinant Affinity Reagent Specific for a Phosphoepitope of Akt1. Int. J. Mol. Sci. 2018, 19, 3305.

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