Figure 2
Workflow of the present study. (a) Design of the libraries A (left) and B (right), based on the VH-FR1-targeted site-directed randomization and insertion, respectively. The library A was composed of scFv sequences, whose codons for amino acids at the positions 1–3, 5–7, 9, and 10 were degenerated to encode 2–6 kinds of predefined residues as indicated. The library B involved the scFv sequences, in which extra 1–6 amino acid residues were inserted between the positions 6 and 7 using the (NNS)n degenerated codons (n = 1–6): this was divided into three sublibraries slB-1–3, slB-4/5, and slB-6. (b) Screening of the libraries with CAP or CAP/ORD system. E. coli TG1 cells were transformed with one of the scFv libraries (A, slB-1–3, slB-4/5, or slB-6) to generate bacterial libraries with similar transformant numbers (0.38–5.1 × 106 cfu), which were each grown on agar plates. Among the resulting colonies, 4,700 were randomly selected and subjected to CAP using 50 microplates: for each plate, 94 microwells were used for inoculating colonies and 2 for background without colonies but with KM13 helper phage. Top 40 scFv-phage clones that showed higher RLU (> 100,000 RLU) were analyzed for binding to cortisol with competitive ELISA7. For slB-1–3, top 188 clones were also subjected to ORD. Then, 40 (after CAP) or 24 (after CAP/ORD) scFv-phages that afforded higher sensitivity (as the midpoint) than wt-scFv were converted to the soluble-form scFvs for examining the affinities. The pie charts show the distribution of luminescent signal (RLU) detected for each single microwell in CAP screening. The background RLU (mean ± SD; n = 100) of each library was varied between microplates as follows: 2758 ± 1909 (A), 3329 ± 3211 (slB-1–3), 507 ± 928 (slB-4/5), 679 ± 2500 (slB-6). (c) Summary of CAP system7. Bacterial clones are individually cultured in microwells containing the helper phage. Therein, each clone propagates and produces scFv-phages without competing with different clones. Antigen-specific scFv-phages bind the pre-immobilized antigen and are detected with a bioluminescence assay using an in-house-prepared fusion protein combining anti-M13-phage scFv and Gaussia luciferase.
