Sf 900 2 serum free medium

The method for production of a Bac M2 was as described in a previously published report [10 (link)]. Bac M2 was grown in Spodoptera frugiperda 9 (Sf-9) insect cells (Invitrogen, Carlsbad, CA, USA) at 28℃ using SF-900 II serum-free medium (Gibco BRL, Rockville, MD, USA). In order to obtain Bac M2, the final culture supernatants of infected Sf-9 cells were collected, treated with sucrose cushion (25% w/w sucrose in 5 mM NaCl, 10 mM EDTA in phosphate buffered saline [PBS]), and centrifuged at 24,000 rpm for 75 minutes at 4℃. The pellets were resuspended in 0.25 M sucrose and stored in LN₂. The viral titer was determined by plaque assay using Sf-9 cells according to the manufacturer's protocol (Invitrogen).

We used the same procedure as described elsewhere (Pauchet et al. 2020 (link)). Briefly, open reading frames (ORFs) of GH5_2, excluding the stop codon, were amplified by polymerase chain reaction (PCR) using RACE-ready cDNA generated in our previous study (Shin et al. 2021 ). A Kozak sequence was added at the 5′-end of the PCR product by integrating it into the forward PCR primer. The resulting PCR products were cloned into pIB/V5-His TOPO/TA (Invitrogen, Waltham, MA, USA) in an ORF with a V5-(His)6 epitope at the carboxyl-terminus, and constructs in the correct orientation were selected after colony PCR. For two constructs (RBIC5 and RBIC9), codon-optimized synthetic constructs cloned into pIB/V5-His TOPO/TA were obtained from the company GenScript (Piscataway, NJ, USA). Insect Sf9 cells (Invitrogen) were routinely cultured in SF-900 II serum-free medium (Gibco, Paisley, UK). Cells were transfected in six-well plates using FUGENE HD (Promega, Madison, WI, USA) as the transfection reagent. After 72 h, the culture medium of transfected cells was harvested, and cell debris was removed by centrifugation. Recombinant GH5_2 proteins were recovered by immunoprecipitation using anti-V5 agarose beads (V5-Trap, ChromoTek, Planegg-Martinsried, Germany). After immunoprecipitation, agarose beads were resuspended in 150 µl of double-distilled water.

The HEK293T cell line was used for protein expression. The cells were maintained in DMEM containing 12% FBS. For cell transfection, Polyethylenimine (Polysciences) was used according to the manufacturer’s instructions. The clones used for transfection were constructed in pcDNA3.1/myc-His (–) and pFLAG-CMV-2 or their modified forms. C-terminal tags, such as GST, GFP, Flag-His, and so on, were cloned into pcDNA3.1/myc-His (–) by replacing the myc-His tag. Cultured cells were harvested 24 h after transfection. The Drosophila S2 cells were cultured in Sf-900 II serum-free medium (Gibco) at 28 °C. For protein expression in S2 cells, the pUAST expression vector was used. mCherry and other tags were cloned into the C terminus between the Kpn1 and Xba1 sites. The expression plasmids were transfected together with actin-GAL4 by Cellfectin II (Invitrogen) and PLUS reagent (Invitrogen). S2 cells were harvested 48 h after transfection.