2.8 Construction of mock library and enrichment tracking
The pCT3CBN yeast expression vector containing the atezolizumab scFv
construct was modified to include a FLAG-tag (DYKDDDDK), replacing the
original C-terminal myc tag. On day 1, a naïve synthetic scFv
library22 with a diversity of approximately
1×109 was thawed and grown overnight in SD-CAA.
Concurrently, competent yeast were electroporated with FLAG-tagged
atezolizumab scFv and grown in SD-CAA for 2 days. On day 2, the naïve
scFv library was passaged in SD-CAA to an O.D. of 1, and on day 3 the
library was induced for 2 days in SG-CAA. Also on day 3, atezolizumab
scFv-expressing yeast were induced in SG-CAA and incubated for 2 days.
On day 5, a mock library was constructed by adding atezolizumab
scFv-expressing yeast into the naïve library at a dilution of 1:1000.
Rounds of selections were carried out by both biofloating and biopanning
enrichment protocols, as described below. Atezolizumab scFv-expressing
yeast enrichment was analyzed by staining the yeast population after
each round of selection using anti-cmyc Alexa647 antibody (Cell
Signaling Technology, clone 9B11) (1:50 dilution) and anti-FLAG DyLight
488 (20 µg/mL) (Thermo Fisher Scientific). FLAG detection was used to
quantify the atezolizumab scFv-displaying yeast population and cmyc
detection was used to identify expressing yeast from the naïve scFv
library.