Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

Abstract

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe the application of this novel label-free technique to study the interaction of human EAG1 (hEAG1) channel proteins with the small molecule PIP2. hEAG1 channel has been recognized as potential therapeutic target because of its aberrant overexpression in cancers and a few gain-of-function mutations involved in some types of neurological diseases. We purified hEAG1 channel proteins from a mammalian stable expression system and measured the interaction with PIP2 by BLI. The successful measurement of the kinetics of binding between hEAG1 protein and PIP2 demonstrates that the BLI assay is a potential high-throughput approach used for novel small-molecule ligand screening in ion channel pharmacology.

A New Gene SCY3 Homologous to Scygonadin Showing Antibacterial Activity and a Potential Role in the Sperm Acrosome Reaction of Scylla paramamosain

“…The rSCY3, rSCY4, and rSCY5 significantly increased the induced AR ratio, as well rSCY2, indicating that the SCYs could promote the sperm AR. In addition, the BLI assay is emerging as a novel label-free methodology for detecting protein–small molecule interactions by immobilizing a tiny amount of protein sample on the surfaces of biosensor and measuring the optical changing signals [ 64 ]. Additionally, the analysis of recombinant human erythropoietin (rh-Epo)/liposome interactions [ 65 ] showed that small molecule inhibitors (SMI) bound to Tissue transglutaminase (TG2) potently inhibited cancer cell adhesion [ 66 ].…”

Crucial role of Ca2+/CN signalling pathway in the antifungal activity of disenecioyl‐cis‐khellactone against Botrytis cinerea

“…Similarly, Gst empty vector was obtained using the same experimental steps as earlier. These experiments were according to the procedures as previously reported 33,34 …”

“…The interaction between cnA protein and DK was detected by using the ForteBio Octet Red system (ForteBio, USA) as the procedures previously described in the literature 34–36 . The GST‐tagged cnA protein and the empty GST protein were immobilized onto Super Streptavidin (SSA) Biosensors (Genemore PNG‐MM‐IGT), and the captured biosensors were individually immersed in the wells containing different concentrations of DK (100, 50, 25, 12.5, 6.25, 3.125 μmol L −1 ).…”

“…The interaction between cnA protein and DK was detected by using the ForteBio Octet Red system (ForteBio, USA) as the procedures previously described in the literature. [34][35][36] The GST-tagged cnA protein and the empty GST protein were immobilized onto Super Streptavidin (SSA) Biosensors (Genemore PNG-MM-IGT), and the captured biosensors were individually immersed in the wells containing different concentrations of DK (100, 50, 25, 12.5, 6.25, 3.125 μmol L −1 ). The association at each DK concentration was monitored for 40 s, while the dissociation was detected for 30 s. The sensors loaded the Gst-tagged cnA protein without DK and the sensors loaded Gst empty vector with various concentrations of DK (100, 50, 25, 12.5, 6.25, 3.125 μmol L −1 ) were used as controls to correct baseline drifts.…”

The combination of salvianolic acid A with latamoxef completely protects mice against lethal pneumonia caused by methicillin-resistant Staphylococcus aureus

“…The kinetics of the binding of Sal A to SrtA were determined by a biolayer interferometry (BLI) assay using the Octet RED96 system (ForteBio, Inc., Menlo Park, CA) as described previously [24]. Ni-NTA sensors were used to bind the His-tag SrtA protein.…”