A fluorescence resonance energy transfer-based binding assay for characterizing kinase inhibitors: important role for C-terminal biotin tagging of the kinase.
Novel biochemical strategies are needed to identify the next generation of protein kinase inhibitors. One promising new assay format is a competition binding approach that employs time-resolved fluorescence resonance energy transfer (TR-FRET). In this assay, a FRET donor is bound to the kinase via a purification tag, whereas a FRET acceptor is bound via a tracer-labeled inhibitor. Displacement of the tracer by an unlabeled inhibitor eliminates FRET between the fluorophores and provides a readout on binding. Although promising, this technique has so far been limited in applicability in part by a lack of signal strength is some cases and also by an inability to predict whether a particular tagging strategy will show robust FRET. In this work, we sought to better understand the factors that give rise to a strong FRET signal in this assay. We determined the magnitude of FRET for several tyrosine kinases using different purification tags (biotin, glutathione S-transferase [GST], and His) placed at either the N terminus or C terminus of the kinase. It was observed that coupling the FRET acceptor to the kinase C terminus using a biotin/streptavidin interaction resulted in the greatest increase in FRET. Specifically, for multiple kinases, the signal/background ratio was at least 3-fold better using C-terminal biotinylation compared with tagging at the N terminus using a His/anti-His antibody or GST/anti-GST antibody interaction. In one case, the FRET signal using C-terminal biotin tagging was more than 150-fold over background. This strong FRET signal facilitated development of improved inhibitor binding assays that required only tens of picomolar enzyme or tracer-labeled inhibitor. Together, these results indicate that C-terminal biotinylation is a promising tagging strategy for developing an optimal FRET-based competition binding assay for tyrosine kinases.
Kwan
Joyce
J
Department of Inflammation Discovery, Roche Palo Alto, Palo Alto, CA 94304, USA.
Ling
Alden
A
Papp
Eva
E
Shaw
David
D
Bradshaw
J Michael
JM
D000077329:Agammaglobulinaemia Tyrosine Kinase; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005982:Glutathione Transferase / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019809:Streptavidin / Q000378:metabolism
D000906:Antibodies; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D047428:Protein Kinase Inhibitors; D006639:Histidine; D001710:Biotin; D019809:Streptavidin; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase