GSM8787771: presort rep 2; Saccharomyces cerevisiae; OTHER
A YSD vector (pCTCON) containing the APPI gene was isolated from the naïve and sorted libraries by using the USB PrepEase Yeast Plasmid Isolation Kit (Affymetrix, Santa Clara, CA) according to the manufacturer's protocol. A naive library of mutated APPI-3M variants, generated using both site-directed random mutagenesis of the APPI-3M binding loop (residues 11–18, except invariant Cys-14) and error-prone PCR amplification of the entire coding sequence, was displayed on the yeast cell surface and presented to pairs of proteases. Each protease in the pair was labeled with a different fluorescent dye. Flow cytometry sorting was used to screen the library to isolate APPI-3M variants, with enhanced selectivity toward each of the serine proteases (mesotrypsin; KLK6). In each sort, two variant populations were collected inside gates, yielding sorted library populations of protease-selective APPI-3M variants.