WT and R217E mutant of Ci-VSD-1-260 constructs were biotinylated through amine
coupling with NHS-SS-PEG4-Biotin (Thermo Scientific). Phage display selections were
performed using a synthetic antibody library built on the 4D5 Fab scaffold as described
previously (Rizk et al., 2011 (link)). Target protein
concentrations were serially adjusted using 100 nM in the first round and 10 nM and 5 nM
in subsequent rounds to ensure proper stringency. The conformation specific binders were
selected using a competitive selection strategy where 1 μM nonbiotinylated
competitor (such as Ci-VSD WT) was added to the phage library prior to the incubation with
actual selection target (such as Ci-VSD R217E). A single point competitive ELISA step used
to identify positive clones. Fabs were expressed in E. coli strain 55244 in phosphate
depleted media as described previously (Rizk et al.,
2011). Fabs were purified using automated two step protocol employing a custom
build AKTA purification system with 5ml rProteinA Sepharose 4 Fast Flow and 1ml Resource S
column (GE Healthcare) columns. Fractions were verified by SDS-PAGE, pooled and dialyzed
against PBS.