Escherichia coli bl21 de3 plyss cells

A synthetic clone was generated encompassing the nucleotide sequences encoding for EDIII of DENV2 (strain Jamaica/1409/1983, amino acids 296–394, Figure S2) with an N-terminal 6x-histidine tag and tobacco etch virus (TEV) cleavage sequence into pRSET_A (Genewiz, South Plainfield, NJ, USA), and transformed into Escherichia coli BL21(DE3) pLysS cells (Promega, Dane County, WI, USA). Cells were grown in LB media supplemented with 100 µg/mL of ampicillin and 25 µg/mL of chloramphenicol, and expression was induced at an OD₆₀₀ of 0.6 using 0.5 mM of IPTG (isopropyl β-D-1-thiogalactopyranoside, final concentration) for 24 h at 37 °C. Cells were harvested at 3500× g for 30 min, and the pellet was resuspended in PBS (phosphate buffered saline) and frozen at −20 °C.

The cloned pET30a (+) plasmids were transformed in Escherichia coli BL21(DE3)/pLysS cells (Promega, Madison, WI). The transformation reaction mixtures were inoculated in 5 ml of 2× YT medium containing 25 μg/ml of kanamycin and cultured overnight at 37°C in an incubator shaker at 250 rpm. A 5-ml volume of each culture was used to inoculate 1 liter of 2× YT medium containing 25 μg/ml of kanamycin. When the optical density at 600 nm (OD₆₀₀) of bacterial culture reached approximately 0.4 to 0.6, IPTG (isopropyl-β-d-thiogalactopyranoside; final concentration of 0.5 mM) was added to the media and the culture was grown for another 2 to 3 h at 37°C or overnight at 16°C. His-tagged recombinant proteins were purified as described previously (37 (link)). The purified proteins were stored at −80°C for use.

Human wild-type (WT) Ccs1 (UniProtKB—O14618) was cloned into a pAG8H vector containing an inducible lacZ site, N-terminal His₈-tag, and an internal tobacco etch virus (TEV) cleavage site using NarI and SalI restriction sites. Sod1 WT (UniProtKB—P00441) was cloned using NarI and HindIII sites into the same vector. Mutations in Ccs and Sod1 were generated using the Thermo Scientific site-directed mutagenesis kit according to the provided protocol. Escherichia coli BL21 DE3 PLysS cells (purchased from Promega) were transformed and grown at 37 °C in 2xYT medium to an A_600nm of 0.6–0.9 and induced with 3–5 mM IPTG. After an additional 4 h of growth cells were harvested and purified using a His-Trap HP Ni²⁺ affinity column by GE Healthcare. The His₈-tag was removed from the purified protein by digestion at room temperature overnight with TEV protease engineered to contain a non-cleavable His₈-tag. The resulting cleaved His₈-tag as well as the TEV protease were removed from Ccs1 by another Ni²⁺ affinity purification.