Technologies for Proteome-Wide Discovery of Extracellular Host-Pathogen Interactions

Martinez-Martin, Nadia

Over a decade ago, fueled by the recent completion of the human genome, Genentech pioneered a significant effort to identify novel secreted or transmembrane domain-containing proteins, upon careful bioinformatics assessment and high throughput protein purification [68] . These efforts resulted in the generation of a comprehensive human protein library, which was subsequently utilized to develop an extracellular protein microarray platform, consisting of over 1,500 secreted or single-transmembrane domain containing proteins [10] . For the generation of this human protein library, secreted proteins or the ECD of single-transmembrane receptors were fused to different affinity tags and subsequently purified from cell culture supernatants by size-exclusion chromatography. Mammalian cells or baculovirus-insect cells were preferentially used as expression systems, to maximize the likelihood of proper folding and glycosylation of the extracellular protein collection [10, 69] . SDS-PAGE and multiangle laser light scattering were used to analyze noncovalent aggregation and ensure high-quality protein production. Subsequently, the purified proteins were spotted on epoxysilane slides using a NanoPrint Arrayer, and protein immobilization on the microarray was determined by probing the slides with the relevant anti-tag antibodies [10] . To enhance detection of low affinity interactions, a rapid method to assemble bait proteins (whose ECD was expressed as a Fc tag-fusion protein) into multivalent complexes using fluorescently labeled protein A microbeads was developed. Proof-of-concept assays showed high sensitivity for detection of weak ePPIs characterized by micromolar , a minimal off-target binding, and more than 70% true-positive to false-positive detection ratio [10, 69] . Over the years, this extracellular protein microarray has successfully identified counterreceptors for a number of human molecules, providing relevant insights into novel pathways that coordinate a multitude of cell functions [70] [71] [72] .