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. 2013 Jul 10;8(7):e70190. doi: 10.1371/journal.pone.0070190

Figure 4. The construction of a proof of principle mating library and the isolation of high affinity, and high expression antigen binders using Fc-Sed1p.

Figure 4

A) Haploid Pichia pastoris strains containing Fc-Sed1p expression cassette were transformed with a library of Anti-PCSK9 Hc or a library of Anti-PCSK9 Lc. Following mating and selection, diploids were cultured to express full length IgG library. Cultures were labeled using 20nM biotinylated human PCSK9 and APC 635 labeled antihuman Kappa. DyeLight 488 labeled Streptavidin was used to detect biotinylated PCSK9. B) Analysis and enrichment of high affinity anti-PCSK9 binders using three rounds of sequential sorting (S1 > S2 > S3). Known anti-PCSK9 strain co-expressing Fc-Sed1p was labeled as above as a control. C) Sensograms showing binding and dissociation kinetics of rhPCSK9 to immobilized anti-PCSK9 antibodies. Each anti-PCSK9 antibody lead was captured on the chip to ~ 500 RU followed by analyte injections of wild-type human PCSK9 at concentrations ranging from 0.156–2.5nM. Kinetics for the highest PCSK9 concentration (2.5 nM) is depicted for these antibodies.