Isolation and Expression of Anti-BoNT VHHs

The VHH-display phage libraries were panned for binding to ciBoNTA or ciBoNTB targets that were coated onto a well of a 12 well plate. Coating was performed by overnight incubation with one ml of a 5 µg/ml target solution in PBS at 4°C followed by washing with PBS and 2 hrs incubation at 37°C with blocking agent (4% non-fat dried milk powder in PBS). Panning, phage recovery and clone fingerprinting were performed as previously described [29] (link), [30] (link). A total of 192 and 142 VHH clones were identified as strong positives for binding to BoNT/A and BoNT/B respectively based on phage ELISA signals. Of the strong positives, 62 unique DNA fingerprints were identified among the VHHs selected for binding to BoNT/A and 32 for VHHs selected for binding to BoNT/B. DNA sequences of the VHH coding regions was obtained for each phage clone and compared for homologies. Based on this analysis, 12 of the anti-BoNT/A VHHs and 11 anti-BoNT/B were identified as unlikely to have common B cell clonal origins and selected for protein expression.
Expression and purification of VHHs in E. coli as recombinant thioredoxin (Trx) fusion proteins containing hexahistidine was performed as previously described [30] (link). For heterodimers, DNA encoding two different VHHs were joined in frame downstream of Trx and separated by DNA encoding a 15 amino acid flexible spacer ((GGGGS)₃). All VHHs were expressed with a carboxyl terminal E-tag epitope. Some expression constructions were engineered to contain a second copy of the E-tag by introducing the coding DNA in frame between the Trx and VHH domains. An example of a Trx fusion to a VHH heterodimer with two E-tags is shown in Figure S1C. A third E-tag was introduced in frame within the DNA encoding the flexible spacer of the heterodimer containing ciA-D12 and ciA-F12 to create a triple tagged heterodimer (D12/F12(3E)).