Fig. 1
Yeast Surface Display (YSD) plasmids construction and maps. Yeast multicopy plasmid pYES2 was sequentially modified by the insertion of the MFα1 sequence fused to the FLAG tag (1), followed by the cloning of the cell wall anchor sequence (SAG1-Cter) (2). In the resulting vector, between MFα1-FLAG and SAG1-Cter was (in-frame) cloned the coding sequence of either yeast metallothionein (yCup1), GFP protein, or exa-Histidine peptide (6xHis), generating pGAL-YSD plasmids series (3). The substitution of the pGAL1 (inducible) with the pGAP (constitutive) promoter originated the pGAP-YSD plasmids, constitutively expressing the chimeric proteins in yeast cells (4). Then, the nutritional yeast recessive URA3 marker was substituted by the dominant NrsR marker, to use pYSD plasmids in natural strains (5). Two additional copies of yCup1 protein were cloned in the pYSD-yCup1 plasmid to generate the chimeric protein with 3 repeats of yCup1 fused as tandem (6). Plasmid functional elements and their modifications are indicated by colors. 2µ ori: yeast replication; pUC ori: E.coli replication; AmpR: E.coli selection marker; CYCter: yeast transcription termination sequence. The name of each plasmid is reported, accordingly to Supplementary Table ST2. Image was created with BioRender.com