Fig. 2.

Schematic representation of the screening set-up. We created a plasmid library by making different combinations of three DNA fragments: five different promoters, six coding regions for our selected proteins and two types of vector backbones, all containing the cyc1-terminator and either LEU2 or URA3 marker for recombinant selection. Plasmids from the library were transformed into the two background strains, wild-type and Δopi1, both having a single-copy of light and heavy chain genes under GAL1-promoter integrated into the HIS3 locus of their genome. Strains were inoculated from solid media to 1 ml of liquid media in 96-deep well plates, in which the strains were precultured for 21 h at 30 °C. The preculture was divided into three plates with fresh media and grown for 5.5 h at 30 °C before antibody expression was induced with 0.5 or 2 % galactose. Antibody was expressed for 24 h at the specified temperatures (20, 25 and 30 °C). Final OD₆₀₀ of cultures was measured. Antibody titers were determined from cleared culture supernatants using two technical replicates with a high-throughput (HTP) ELISA on an automated liquid handling station. Each strain was analyzed in three independent biological replicates