Branched multimeric peptides as affinity reagents for detection of α-Klotho protein

-Klotho is a protein associated with aging that is expressed in the kidney, parathyroid gland, and choroid plexus. As a transmembrane protein, it acts as an essential co-receptor with the fibroblast growth factor 23 receptor complex to regulate serum phosphate and vitamin D levels. -Klotho has an extracellular domain that can be cleaved, released and circulated in the blood stream as a soluble form. Decreased levels of -Klotho are an indication of chronic kidney disease and other age-associated diseases. Detecting or labeling transmembrane and soluble -Klotho is a longstanding challenge that has impeded the in-depth understanding of its role. Here we describe branched multimeric peptides that recognize -Klotho with high affinity and selectivity in the biological milieu. The branched peptides are prepared in a single-shot synthesis by parallel automated fast-flow synthesis in under one hour. The branched -Klotho-binding peptides show improvement in affinity relative to the monomeric versions and can be used to label Klotho for live imaging in kidney cells. Our results demonstrate the potential of automated flow technology to deliver peptide-based reagents with complex architecture and improved affinity for the selective binding of target proteins in physiological settings.