Ja 10

Low passage Chinese hamster ovary (CHO) cell lines Pro5 and Lec2 were cultured as monolayers in MEM-α (ATCC) with 10% FBS (fetal bovine serum) and 1% antibiotic-antimycotic in a 5% CO₂ 37 °C incubator. For cell binding assays, the CHO cells were detached from plates by addition of EDTA, pelleted, resuspended in MEM-α to 5 × 10⁵ cells/mL, pre-chilled for 30 min at 4 °C, and aliquoted to 500 µL fractions. Each tube of cells was then incubated with the fluorescently labeled VLPs at a MOI of 10⁶ under constant rotation for 3–4 h at 4 °C (protected from light). Following the incubation, the cells were pelleted at 2000 rpm (Beckman JA-10) for 10 min and the supernatant discarded. Unbound VLPs were removed by washing the cells with 300 µL ice-cold 1× PBS, followed by centrifugation. Pellets were resuspended in 300 µL 1× PBS and analyzed utilizing a FACS Canto (BD, Franklin Lakes, NJ, USA). Cells without added fluorescent-labeled capsids were used as baseline to determine the percentage of fluorescent cells for the other samples. All experiments were conducted in triplicate. The FSC Express5 software suite (De Novo Software, Pasadena, CA, USA) was used to analyze the raw data.

E. coli K12 MG1655 cells were grown for 12 h at 37 °C in 500 mL of LB broth. Cells were pelleted by centrifugation at 8000 rpm for 10 min using the JA10 Beckman Coulter rotor at 4 °C. The cell pellet was washed twice with 20 mM HEPES, 100 mM NH₄Cl, pH7.4 and dissolved in lysis buffer (20 mM HEPES, 100 mM NH₄Cl, 10.5 mM Mg acetate, 0.5 mM EDTA, 5 mM 2-mercaptoethanol, 3 mM PMSF and Roche cOmplete protease inhibitor tables 1/10 mL). The cells were lysed using Emulsifier EmulsiFlex-C3 at 10 000 PSI for 6 cycles at 4 °C. The lysate was collected and centrifuged at 15 000 rpm for 45 min in a Beckman Coulter JA-20 rotor. The protein concentration was determined by Bradford assay using BSA as a standard. The cell lysate was aliquoted and diluted with cross-linking buffer (25 mM HEPES-NH₄OH, 100 mM NH₄Cl, 10.5 mM Mg acetate, pH 7.4@20 °C).

E. coli CH2016 cells^{60 (link)} carrying plasmid pCH12802 were grown in LB medium supplemented with 100 μg/mL ampicillin, and CdiI^EC3006-His₆ production was induced with 100 µM IPTG. After 3 h, cells were collected by centrifugation at 4420 × g for 15 min in a Beckman JA-10 rotor and frozen at –80 °C. Cells were resuspended in ice-cold lysis buffer containing 2 μM leupeptin, 2 μM peptstatin, 100 μM phenylmethylsulfonyl fluoride (PMSF), 5 μM tosyl-l-lysyl chloromethane HCl, then broken by passage through a French pressure cell. Cell lysates were clarified by centrifugation at 14,600 × g in a Beckmann JA-20 rotor for 1 h at 4 °C. The supernatant was adjusted to 5 mM MgCl₂ and 500 μg of DNase I was added to digest DNA on ice for 20 min. The lysate was applied to a Ni²⁺-loaded Chelating Sepharose Fast Flow (GE Healthcare) column pre-equilibrated with lysis buffer. The column was then washed with six column volumes of lysis buffer. His₆-tagged CdiI^EC3006 was then eluted with lysis buffer supplemented with 250 mM imidazole. The eluate was concentrated to <2.5 mL using an Amicon Ultra-15 10 kDa molecular weight cut-off centrifugal concentrator and applied to a PD-10 desalting column (GE Healthcare) equilibrated with 50 mM NaPO₄ (pH 6.5). Purified CdiI^EC3006-His₆ was quantified by absorbance at 280 nm (21,890 cm⁻¹ M⁻¹).