S1 domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen

Makadiya, Niraj; Brownlie, Robert; van den Hurk, Jan; Berube, Nathalie; Allan, Brenda; Gerdts, Volker; Zakhartchouk, Alexander

Human embryo kidney (HEK) 293 T (ATCC CRL-1573) and Vero 76 (ATCC CRL-1587) cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 50 μg/mL gentamycin and 10 % fetal bovine serum. PichiaPink Strain 1 (Invitrogen) was grown in YPD medium. Insect Sf9 cells (Gibco) were grown in Sf-900 serum-free medium (ThermoFisher Scientific). Expression of recombinant S1 protein in yeast cells Yeast codon optimized PEDV S1 gene (coding animo acids 1-734) with the 3'-end polyhistidine coding sequence was synthesized by GenScript. Synthetic S1 gene was digested with restriction enzymes MlyI-KpnI and cloned into the pPinkα-HC vector (ThermoFisher Scientific) into StuI-KpnI sites. Expression of the recombinant protein was performed as recommended by manufacturers (ThermoFisher Scientific). Briefly, PichiaPink strain 1 (ade2 − ) was electroporated with pPinkα-S1 linearized plasmid DNA and plated on PAD (Pichia Adenine Dropout) agar plates for selecting transformants. After incubation at 30°C for 5 days, white colonies were screened for expression of S1. Recombinant yeast cells were grown in buffered glycerol-complex medium (BMGY) and induction was performed in buffered methanol-complex medium (BMMY) with 0.5 % methanol. To prevent non-specific cleavage of S1 protein by yeast cell proteases, aprotinin and phenylmethylsulfonyl fluoride (PMSF) were added during the induction phase at 1 μg/mL each. Yeast cells were grown at 30°C for 4 days and centrifuged at 3000 g for 5 min at room temperature and supernatant was collected for protein purification.