Favorprep gel purification kit

The method to study the effect of peptide treatment on in vitro expression of protein was adapted from previous study (Taniguchi et al., 2016 (link)). RTS^TM 100 E. coli HY Kit (biotechrabbit) was used as a cell-free rapid translation system (RTS) to express green fluorescent protein (GFP) with or without peptide treatment. Streptomycin (Sigma-Aldrich), an antibiotic functioning as an inhibitor of bacterial translation, was used as a positive control. Reaction mixture was prepared as described in the product manual. For the negative control, the remaining 10 μl was topped up with nuclease-free water while for the other reactions, the 10 μl consists of 5 μg GFP mRNA and either nuclease-free water or the indicated treatment (100 μM Pen, Pen-BR, Pen-RRR, CapM2 or 10 μM Streptomycin). Reaction was incubated at 30°C for 6 h before being analyzed with Western blot for the protein level of GFP. To obtain GFP mRNA, control GFP expression vector was first linearized using ApaLI restriction enzyme (New England Biolabs) and then separated via agarose gel electrophoresis. Fragment containing the linearized GFP expression vector was retrieved using FavorPrep GEL Purification Kit (FAVORGEN Biotech Corp.) and subsequently used as the template for MEGAscript^TM T7 Transcription Kit (Thermo Fisher Scientific) to generate GFP mRNA.

For plasmid constructions, P. berghei merozoite surface protein 8 (MSP-8, codon-optimized), merozoite surface protein 9 (MSP-9) and influenza matrix protein 1 (M1) genes were cloned, as described previously [15 (link),16 (link),25 (link)]. Codon-optimized P. berghei rhoptry-associated protein 1 (RAP1) gene was acquired from GenScript (Piscataway, NJ, USA).
The recombinant plasmid was transformed into DH10Bac competent cell. Colonies were screened and bacmid DNA from successful clonal construct was extracted using FavorPrep Gel Purification Kit (Favorgen, Cheshire, UK). MSP-8, MSP-9, RAP1, or influenza M1-expressing recombinant baculoviruses (rBVs) were prepared as previously described [26 (link),27 (link)]. Three different VLPs, each expressing one of MSP-8, MSP-9, or RAP1 antigen were generated along with influenza M1 core protein as described previously [26 (link),27 (link)]. After co-culturing Sf9 cells with the rBVs expressing the antigens of interest, culture media were collected at 3 days post-infection (dpi). Cells were pelleted by centrifuging for 1 h, 4 °C, 4800× g. VLPs present within the supernatants were purified and stored at 4 °C as described elsewhere [26 (link),27 (link)].

The endopolygalacturonase genes (Pg1, Pg5), exopolygalacturonase genes (Pgx1, Pgx4), translation elongation factor 1-α (TEF1-α), and intergenic spacer (IGS) region of rDNA were amplified in a polymerase chain reaction (PCR) using the gene-specific primer pairs given in Supplementary Table S1. The PCR analysis was conducted in a thermal cycler (VeritiTM 96 wells, Applied Biosystems) with a reaction volume (25 μL each) comprised of 15 ng of fungal gDNA; a primer pair (10 mM each), 12.5 μL Phusion High-Fidelity PCR Master Mix with HF Buffer, following a thermal program; 95 °C for 3 min, 35 cycles with denaturation at 95 °C for 50 s; annealing temperature (Supplementary Table S1) for 35 s, extension at 72 °C for 90 s, and final extension at 72 °C for 8 min. No template control (NTC) was run in all reactions as a negative control. PCR products were electrophoresed on high-resolution agarose gel (0.6%); required amplicons were eluted from the gel using a FavorPrep Gel purification kit (Favorgen Biotech Corporation, Taiwan), and sent to Eurofins Genomics DNA sequencing services, USA. All generated sequences were trimmed (BioEdit software) and subjected to the Blastn tool for searching their homology before depositing in the GenBank to obtain accession numbers (Supplementary Table S3).