Engineered Glycan-Binding Proteins for Recognition of the Thomsen–Friedenreich Antigen and Structurally Related Disaccharides

Abstract

Glycan-binding proteins (GBPs) are widely used reagents for basic research and clinical applications. These reagents allow for the identification and manipulation of glycan determinants without specialized equipment or time-consuming experimental methods. Existing GBPs, mainly antibodies and lectins, are limited, and discovery or creation of reagents with novel specificities is time consuming and difficult. Here, we detail the generation of GBPs from a small, hyper-thermostable DNAbinding protein by directed evolution. Yeast surface display of a variable library of rcSso7d proteins was screened to find variants with selectivity toward the cancer-associated glycan Galβ1− 3GalNAcα or Thomsen−Friedenreich antigen and various relevant disaccharides. Characterization of these proteins shows them to have specificities and affinities on par with currently available lectins. The proteins can be readily functionalized with fluorophores or biotin using sortase-mediated ligation to create reagents that prove useful for glycoprotein blotting and cell staining applications. The presented methods for the development of GBPs toward specific saccharides of interest will have great impact on both biomedical and glycobiological research.