University of British Columbia Uses the Octet RED96e System to Quickly Determine Interaction Kinetics

Biotinylated Version of the Peptidisc Peptide
 

University of British Columbia

James Saville, Lucy Troman, Franck Duong Van Hoa

8-Channel Octet Systems: Octet RED96e

The Challenge

Membrane proteins (MP) constitute only 30% of our proteins, yet comprise over 60% of current therapeutic targets. This overrepresentation of membrane proteins as drug targets is because they are easily accessible on the outside of cells and play crucial roles in the uptake of nutrients, cell communication, and generally allow the cell to interact with its environment. Despite the obvious fundamental and medical relevance, membrane proteins are severely underrepresented in structural and interaction databases largely due to the inherent insolubility of membrane proteins in aqueous solution, a pre-requisite for nearly all biochemical analyses. This solubility problem is a major barrier to MP characterization and exploitation as therapeutic targets.

The Solution

Members of the Duong laboratory, at the University of British Columbia, recently developed a membrane mimetic, called the peptidisc, that replicates the native membrane environment, while keeping membrane proteins soluble in solution. The peptidisc is formed when multiple copies of an amphipathic peptide bind onto the hydrophobic surface of the target membrane protein. Stable reconstitution into peptidisc occurs upon detergent removal, entrapping both lipids and membrane proteins into water-soluble particles. The peptidisc formulation is guided by the size and topology of the target membrane protein itself, and therefore occurs in a spontaneous manner, requiring little optimization.

To analyze protein-ligand interactions of peptidisc-stabilized membrane proteins, using the established streptavidin-based binding assays available on the Octet RED96e, the lab introduced a biotinylated version of the peptidisc peptide. The biotin functional groups allow attachment of the peptidisc particles onto streptavidin-coated tips via the disc. These ligand-bound tips were incubated in a solution containing a binding partner (the analyte) and then passed into buffer where the dissociation of the analyte and ligand was measured. This automized measurement was left to run on the Octet at the end of the day, and the resulting data analyzed in 10 minutes the following morning.

Octet System Red96e

8-Channel Octet Systems: Octet RED96e

Products Used

ForteBio’s 8-channel Octet systems perform quantitation of 96 samples in 32 minutes and kinetic screening of 64 samples in 1.5 hours. Analysis can be done using a single channel or up to eight channels, enabling flexibility in sample throughput based on need. An optional microplate evaporation cover that minimizes losses in sample volume is available with the Octet RED96e.

The Results

Using the biotinylated peptidisc peptide and the Octet RED96e system, the Duong lab determined the binding kinetics of the bacterial membrane receptor (FhuA) and the antimicrobial peptide Colicin M. The devised workflow including the Octet RED96e allowed them to go from grown cells, to measured interaction within 24 hours.

The real-time readout of interaction kinetics produced by the Octet allowed the Duong laboratory at the University of British Columbia to immediately see if their assay was set-up correctly and if they were detecting their interaction.

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