Top10 cells

Stbl3 (Thermo Fisher Scientific, C737303) and TOP10 cells (Thermo Fisher Scientific, C404010) were transformed with pUC19 and pBAD derived plasmids respectively. Cells were grown overnight in LB with the appropriate antibiotic to stationary phase. For liquid culture experiments, 3 μL was used to inoculate 150 μL cultures in clear 96-well plates. Plates were sealed with clear optical film and two holes were punched for aeration using a 28 gauge needle. Plates were incubated in a Synergy Neo2 plate reader (BioTek) at the indicated temperature with constant orbital shaking and the optical density at 600 nm read every 5 minutes. Plate-based growth assays were performed by normalizing the input density of overnight cultures and performing 10-fold dilutions. 5 μL of each dilution was dropped onto agar plates and grown at the indicated temperature for 16 hours. Plates were imaged using a Chemi-Doc (Bio-Rad).

pcDNA3.1-HADHA, pcDNA3.1-HADHB, pcDNA3.1-myc-his and pcDNA3.1-viperin have been previously described [10 (link), 11 (link)]. All primers for making mutants were synthesized by the Keck facility at Yale University. Deletion and point mutants were made from pcDNA3.1-viperin via PCR using Turbo-Pfu (NEB). PCR products were digested with DpnI (NEB) for 5h at 37°C before being transformed into DH5α or Top10 cells (Thermo Fisher Scientific). Viperin was cloned into pRetroX (Clontech) by PCR using Taq-HiFi (NEB) and digested with restriction enzymes (NEB), ligated using T4 DNA ligase (NEB) into the digested vector backbone, and transformed into Stbl3 cells (Thermo Fisher Scientific). pCMV-FLAG-MAVS was a kind gift from the laboratory of Dr. Akiko Iwasaki. pCMV-SPORT6-ΔtmMAVS was a kind gift from the laboratory of Dr. Yorgo Mordis. pcDNA3.1-MDA5 and pcDNA3.1-RIGI were a kind gift from Dr. Shu Zhu in the laboratory of Dr. Richard Flavell. Plasmids were purified using Qiagen miniprep kits or kits from Origene and Zymo Research.

Bm-UGT was expressed as a Renilla reniformis luciferase (Ruc) fusion protein by cloning the Bm-UGT coding sequence in pREN2 (Genscript). The Bm-UGT signal sequence as predicted by signalP was removed prior to synthesis. Plasmid encoding the fusion protein was used to transformed TOP10 cells (Thermo Fischer) and plasmid DNA was obtained from colonies selected on kanamycin (50 μg/ml) as per the manufacturer’s guidelines (Qiagen Midi-Prep). 293F cells grown in 293 Freestyle Medium as suspension cultures were transfected with 30 μg of Bm-UGT-Ruc plasmid, at a final concentration of 1 μg per 1 x 10⁶ cells (Thermo Fischer Sceintific) per mL, and cultured at 37°C with 8%CO₂ on a rotary shaker at 125 rpm. After 72hrs, the cells were pelleted and sonicated in LIPS lysis buffer (20 mM Tris pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 1% TritonX-100, 50% glycerol, protease inhibitors (Mini from Roche)). The lysate was centrifuged to remove cellular debris and supernatant containing the Bm-UGT-Ruc fusion proteins were stored at -80°C for later use.

The Bma-LAD-2-Renilla reniformis luciferase (Ruc) construct was inserted into a pREN2 vector by GenScript. The predicted signal sequence was removed prior to gene synthesis. The vector was cloned into TOP10 cells (Thermo Fisher Scientific) and amplified on agarose plates with kanamycin at 50 μg/mL. Plasmid DNA was isolated and purified using a Miniprep kit (Qiagen) per the manufacturer’s guidelines. 293F cells (Thermo Fisher Scientific) were transfected with 30 μg of Bma-LAD-2 plasmid at a concentration of 1 × 10⁶ cells per mL. The 293F cells were collected 72 h later and homogenized. The lysate was stored at −80°C for later use.

Based on the sequencing analysis, representative clones were selected and reformatted to rabbit/human chimeric IgG1 isotype format comprised of rabbit variable (VH and VL) and human constant domains of IgG1/kappa isotype. Construction of chimeric expression vectors was carried out by cloning of the rabbit heavy and light chain variable regions into separate vectors containing human constant domains, pHuG1 (IgG1 isotype) and pHuK (kappa), respectively, using ligase-independent cloning and transformed into chemically competent TOP10 cells (C404010, ThermoFisher). Several clones were isolated, prepped using the QIAprep Spin Miniprep Kit (27103, QIAGEN), and sequences were verified by Sanger sequencing. To produce chimeric IgG candidates, a pair of plasmids encoding heavy and light chains for each clone was cotransfected into Expi293 suspension cells using ExpiFectamine293 reagent (The Expi293 Expression System Kit, Invitrogen—manufactures standard instructions). After 5 days of cultivation, culture supernatants were collected, and IgG concentration was quantified by OctetRED384 (ForteBio). Supernatants from large-scale cultures were purified by standard antibody purification methods using Protein A or G affinity chromatography followed by size-exclusion chromatography. Concentrations of the purified antibodies were measured by absorbance at 280 nm.