Figure 1.
FACS analysis of the different YSD APPI libraries, showing expression and KLK6 binding. A, YSD vector (pCTCON-2) is aligned with the general scheme of the insert: the insert gene consists of the APPI gene, which is flanked by two restriction sites (BamHI and NheI), followed by a linker sequence (LPDKPLAFQDPS) on the 3′ end, along with two pCTCON homologous sequences. B, APPI is displayed on the yeast cell surface as a translational fusion to Aga2p, which is linked to Aga1p by two disulfide bonds. Surface expression is detected by using fluorescence-labeled antibodies binding to the C terminus of the c-Myc epitope tag, whereas target binding is detected using fluorescence-labeled KLK6, via FACS. C, 3D structure of APPI (PDB code 1ZJD). D, expression of APPI and the binding of 10 nm labeled KLK6 were determined in a YSD system for an APPIWT clone, for a library of mutants based on APPIWT, and for a library of mutants based on APPIG17M,I18F,F34V.