Erythrocyte specific VLRB-YSD library enrichment strategies

YSD libraries were enriched for Type O erythrocyte specific VLRBs using two different methods. The cell surface proteins on red blood cells were biotinylated according to the manufacturer’s instructions using EZ-Link^TM Sulfo-NHS-LC-Biotin (Thermo-Fisher – Pierce). The first method of enrichment involved co-incubating induced YSD libraries with the intact biotinylated erythrocytes for 1 h at room temperature on rotation. The yeast bound to the labeled erythrocytes were then captured on streptavidin labeled magnetic beads (MACS sorting) and eluted directly into SD-CAA media and grown overnight. This library was further enriched by FACS using the same approach. The second method was to co-incubate the induced yeast with the biotin-labeled lysed red blood cells. The success of the MACS and FACS enrichment was monitored by flow cytometry using the anti-Myc 488 and SA-PE secondary reagents. The enriched libraries were transferred onto SD-CAA agar plates in order to sequence and characterize individual monoclonal VLRB clones.