Figure 1 | Scientific Reports

Figure 1

From: Rapid and sensitive large-scale screening of low affinity extracellular receptor protein interactions by using reaction induced inhibition of Gaussia luciferase

Figure 1

Design and expression of prey constructs with different reporter enzymes. (a) Schematic diagrams of the protein constructs used in this study. Prey proteins consisting of the extracellular region of a protein of interest (Gene A) are fused at their C-terminus to the proteins tags: rat Cd4(d3 + 4) and the rat cartilage oligomeric matrix protein (COMP) followed by either five repeating units of luciferase, HRP, or beta-lactamase enzymes. Baits are expressed as monomers and consist of ectodomains of a protein of interest (Gene B) fused to the same rat Cd4(d3 + 4) tag but additionally containing a peptide sequence that is recognised by the enzyme BirA for the covalent addition of a single biotin molecule. Both baits and preys contain a terminal 6-his tag for purification. (b) Schematic representation of the AVEXIS assay involving a soluble highly avid pentameric enzyme-tagged prey protein and biotinylated bait protein immobilised on a streptavidin-coated microtitre plate. (c) Pentameric preys containing the HRP enzyme were expressed at low levels. Rat Cd200 prey constructs containing the named enzyme reporters were expressed by transient transfection of HEK293 cells and the secreted protein yield quantified after nickel affinity purification. Data points are values for three independent transfections and bars represent the means. BLac = beta-lactamase prey; GLuc = Gaussia luciferase prey; NanoLuc = Nano luciferase prey.

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