Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Abstract

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes 1 , but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives < 1 second) precluding their detection with many commonly-used high throughput methods 2 .Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t 1/2 ≤ 0.1 sec) with a low false positive rate 3 . The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.The recombinant protein libraries are produced using a convenient and high-level mammalian expression system 4

From molecules to mating: Rapid evolution and biochemical studies of reproductive proteins

“…However, significant advancements were recently made towards understanding mammalian membrane fusion. Using a high-throughput ELISA-like screen for membrane protein/ligand interactions [205], folate receptor 4 (FolR4, now renamed “Juno”) was characterized as the egg cell surface receptor for sperm Izumo1 and determined to be critical for membrane fusion. Immunocytochemistry experiments also demonstrated that upon sperm binding, Juno is shed from the oocyte membrane through vesicle formation and exocytosis, acting as a block to polyspermy.…”

RH5–Basigin interaction plays a major role in the host tropism of Plasmodium falciparum

“…AVEXIS assays were performed essentially as described (30,37). Bead-based erythrocyte-binding assays were performed as described (38) using streptavidin-coated Nile red fluorescent 0.4-to 0.6-μm microbeads (Spherotech Inc).…”

Signalling lymphocyte activation molecule family member 9 is found on select subsets of antigen‐presenting cells and promotes resistance to Salmonella infection

“…Soluble mouse SLAMF9 was produced in human HEK293T cells by lentiviral expression using the vector pLenti‐CMV‐GFP‐Puro (a gift from Eric Campeau; Addgene). Soluble SLAMF9 comprised the two immunoglobulin superfamily domains of SLAMF9 fused to rat CD4 domains 3+4 and a 6xHis tag in an AVEXIS‐ready configuration . Protein was expressed in FreeStyle 293 Expression medium and purified by Co 2+ Immobilized Metal Ion Chromatography.…”