Isopropyl β d 1 thiogalactopyranoside (iptg)

The generated phage display affinity maturation libraries were subjected to one round of panning using an affinity capture procedure on recombinant Delta variant RBD, following the protocol described by Viti et al.^[50] Briefly, 120 pmol of biotinylated RBD Delta were immobilized on 60 µL streptavidin‐coated beads (Invitrogen, M‐280). After blocking in 4% milk‐phosphate buffer saline (PBS), 800 µL of scFv displaying phage were added (≈10¹² transforming units of phage per mL; no negative selection was performed prior to the positive selection) and incubated for 1 ½ h. Beads were washed six times with 0.1% Tween 20 in PBS and subsequently six times with PBS. Bound phage were eluted with triethylamine (Sigma) and used to infect fresh E. coli TG‐1. Individual colonies were inoculated in 2xYT media supplemented with 100 µg mL⁻¹ ampicillin. Expression of scFv was induced by adding 1 mm isopropyl β‐d‐1‐thiogalactopyranoside (Applichem) and incubating at 30 °C overnight while shaking (200 rpm). Plates were spun down at 4000 g for 15 min. and supernatants containing soluble antibody fragments were collected. The resulting antibody fragments were screened by ELISA on immobilized RBD WT and Delta. Clones yielding positive ELISA signals for both RBD WT and Delta were sequenced and chosen for further characterization.

All constructs pET-22b-atglcak, pET-16b-atusp, pET-22b-pmppa, pET-22b-blnahk, pET22b-szglmu and pET-22b-pmhas^1-703 were transformed into E. coli BL21 (DE3) via heat shock. The transformed clones were selected on agar plates with lysogeny broth (LB) medium containing ampicillin (100 µg/mL). Clones were further cultivated in baffled shaking flasks. The pre-culture was grown in 20 mL LB medium with ampicillin (37 °C, 120 rpm). The main culture (1 L) with terrific broth was inoculated with 1% (v/v) pre-culture (37 °C, 80 rpm). When the OD₆₀₀ of the main culture reached 0.6–0.8, the expression of the proteins was induced by adding 0.1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG, AppliChem GmbH, Darmstadt, Germany) and the temperature was reduced to 25 °C. After 20 h the cells were centrifuged (7000 rpm, 4 °C), the supernatant was discarded and the cells were stored at −20 °C.

Bacterial cells were cultured in Luria Bertani (LB) broth at 37°C to an optical density of OD₆₀₀ = 0.8. Protein production was induced by the addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (AppliChem GmbH, Germany). Bacteria were further incubated at 16°C for 18 h. Protein extraction and purification were conducted as previously described (Schubert et al., 2012 (link)). HIS-tagged proteins were purified by metal-affinity chromatography using Ni-NTA resins (Qiagen). Protein concentration was estimated by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) and the purity of CCL2 and CCL2-Y92A was examined by SDS-PAGE.

All proline variants were expressed in the E. coli strain Lemo21 (NEB) from a pET-11a-d vector, utilizing the T7 polymerase/T7 lysozyme system^{45 (link)}. Liquid media used for the expression of all variants was 2xYT broth containing 0.1 mg/mL ampicillin (Sigma), 0.03 mg/mL chloramphenicol (Sigma) and 76 µM rhamnose (Sigma). Cultures were grown to a density of A₆₀₀ ~ 0.4–0.8 A.U. and expression was initialized by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) (AppliChem) to a final concentration of 400 µM followed by the addition of sterile 4 M CaCl₂ (Sigma) to give a final concentration of 100 mM and grown at 18 °C and 230 rpm for 20–24 hours. All proline variants were purified to homogeneity as described in^{25 (link)}.

Bacto Yeast Extract, Bacto Agar and Bacto Tryptone were purchased from Becton Dickinson (Basel, Switzerland) and used for the preparation of LB (Luria–Bertani) culture medium. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was obtained from Applichem (Darmstadt, Germany). Polymyxin B sulfate, HEPES [4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid], oxalic acid, MgCl₂, CaCl₂, NaH₂PO₄ and imidazole were from Fluka (Buchs, Switzerland). Ampicillin, bovine serum albumin (BSA), ethylenediaminetetraacetic acid (EDTA), amino acids and BME vitamin mix were obtained from Sigma. The sugars 1,5-anhydro-d-mannitol (AM), n-heptyl α-d-mannopyranoside (HM) and the 4-biphenyl α-d-mannopyranoside (BF) were synthesized as described previously (Pang et al., 2012 ▸ ).