M13ko7

The nanobody gene library was synthesized by GenScript (NJ, USA) following the theoretical design. The genes were flanked with the restriction sites NotI and NcoI for cloning in the pMAC phagemid vector [15 (link)]. After cloning (using 4 μg of both the gene library and pMAC), the recombinant plasmids were transformed by electroporation (voltage 2.5 kV, resistance 200 Ω, capacitance 25 μF) in the E. coli strain SS320, previously transduced with the helper phage M13KO7 (New England Biolabs, Ipswich, MA, USA).
Transformed bacteria were recovered in SOC medium for (i) determining library diversity by seeding serial dilutions in plates containing solid 2xYT medium supplemented with 100 µg/mL ampicillin, and (ii) amplifying the recombinant phage library in 2xYT medium containing 100 μg/mL ampicillin, 50 μg/mL kanamycin, 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) by incubating 20 h at 30 °C and 185 rpm. Phage library was precipitated from the supernatant with 0.2 volumes of a solution containing PEG/NaCl (20% polyethylene glycol 8000 and 2.5 M NaCl) at 4 °C for two hours, and aliquoted in 10% glycerol until further use [44 (link),45 ].

200 mL 2×TY with 2% (w/v) glucose + 100 µg/mL ampicillin was inoculated from an overnight starter culture of E. coli TG1 (Lucigen) transformed with pBAD-DsbA(ss)-SBTI-pIII and grown at 37 °C at 200 RPM until OD₆₀₀ 0.5. Cultures were infected with helper phage at a multiplicity of infection of 10:1 (phage:bacteria) for 45 min at 37 °C at 80 RPM. Bacterial cells were pelleted by centrifugation at 2500 g for 10 min at 4 °C and resuspended in 200 mL induction medium: 2×TY + 0.2% (w/v) L-arabinose + 100 µg/mL ampicillin + 50 µg/mL kanamycin. Phage were grown overnight at 18 °C at 200 RPM.
Phage was precipitated by addition to 5% (w/v) polyethylene glycol 8000 (PEG8000, Thermo Fisher) + 0.5 M NaCl for at least 1 h at 4 °C, centrifuged at 15,000 g at 4 °C, and the supernatant discarded. The phage pellet was resuspended in PBS and centrifuged at 15,000 g at 4 °C to remove bacterial cells. Precipitation was repeated for a total of three rounds. Purified phages were stored at −80 °C in PBS with 15% (v/v) glycerol. Phage stocks were titered in duplicate by quantitative PCR (qPCR) using primers Fwd2 (5′-GTCTGACCTGCCTCAACCTC-3′) and Rev2 (5′-TCACCGGAACCAGAGCCAC-3′) and 2× SensiMix (Bioline) master mix relative to a dilution series of M13KO7 (NEB). qPCR was performed on a Mx3000P qPCR machine (Agilent) and data were analyzed using MxPro qPCR software (Agilent).

Cloning primers with degenerate oligonucleotides were used to introduce histidine mutations randomly in the complementarity‐determining regions (CDRs) of humanized monkey‐derived hu1‐23^[55 (link)
^] and mouse‐derived huE6F6‐1,^[56 (link)
^] and recombinant PCGMT‐expression vectors expressing scFvs were transformed into ER2738 electrocompetent cells (Lucigen, US) and selected with ampicillin (Sangon Biotech, CN).^[57 (link)
^] The helper phage M13KO7 (NEB, US) was used to infect ER2738 cells, and phage‐scFv primary libraries for hu1‐23 and huE6F6‐1 with capacities of 6.7 × 10⁷ and 1.15 × 10⁸, respectively, were constructed. Library biopanning was carried out against a recombinant HBsAg protein (CHO cell‐derived, Wantai Biological Pharmacy Enterprise Co., Ltd., CN) coated on 96‐well ELISA plates, as shown in Figure S1A (Supporting Information). Four rounds of panning were performed, and clones with a stronger binding affinity for HBsAg at pH 7.4 than at pH 6.0 were defined as pH‐dependent positive (Figure S1B, Supporting Information). The genes of the variable heavy chain (vH) and light chain (vL) of the positive clones were sequenced. The heavy chain and light chain variable regions of selected scFv clones were subcloned into pTT5 expression vectors (YouBio, CN) containing the human and murine IgG1 heavy chain and kappa chain constant regions, respectively.^[58 (link)
^]

Recombination phage library was generated in above scFv-TG1 library by the addition of helper phage M13KO7 (NEB, Ipswich, MA, USA), precipitated with polyethylene glycol 8000 (4%, PEG-8000) and NaCl (3%, w/v), and resuspended in 2YT medium and stored at 4 °C. The bio-panning was carried out by adding 10¹² plaque forming units (pfu) of recombinant phages to wells pre-coated with S1 protein followed by incubation at 37 °C for 2 h. The unbound phages were removed by Tween-PBS (PBST), bound phages were eluted with HCl-glycine (pH 2.5, 0.2 M) with end-over-end mixing for 10 min, the lower pH of the eluted phage neutralized with Tris–HCl (pH 9.0) and used to infect the TG1 E. coli. The amplified phages were precipitated and recovered as described above for the next round of selection, and the bio-panning was repeated for three more times. The enrichment of specificity was determined from the input/output ratio of the phage. Phage-infected TG1 cells from each bio-panning were plated onto SOBAG plates to determine the library size. Ten random scFv-phage clones on the plates were selected from the fourth round to identify the positive rate and binding capacity to antigen by PCR and phage ELISA, respectively.