Author: Makadiya, Niraj; Brownlie, Robert; van den Hurk, Jan; Berube, Nathalie; Allan, Brenda; Gerdts, Volker; Zakhartchouk, Alexander
Title: S1 domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen Document date: 2016_4_1
ID: 1r3doeic_5
Snippet: Expression of S1 in yeast cells Initial attempts to express the S1 protein in the bacterial cells were not successful (data not shown), which may be due to problems in processing of the S1 protein in prokaryotic cells. Therefore, we used PichiaPink (Pichia pastoris) yeast cells to express S1 from a synthetic S1 gene codon optimized for yeast and containing a C-terminal histidinetag to aid purification. Initially, the time course was performed for.....
Document: Expression of S1 in yeast cells Initial attempts to express the S1 protein in the bacterial cells were not successful (data not shown), which may be due to problems in processing of the S1 protein in prokaryotic cells. Therefore, we used PichiaPink (Pichia pastoris) yeast cells to express S1 from a synthetic S1 gene codon optimized for yeast and containing a C-terminal histidinetag to aid purification. Initially, the time course was performed for the expression of the S1 protein in the yeast cells over the period of 4 days. Western blot analysis of the cell culture medium of transformed yeast cells resulted in the detection of a specific 35-40 kDa band when probed with anti-his antibody (Fig. 1a) . The observed protein molecular weight was less than the expected 80.9 kDa molecular weight of un-glycosylated S1. This may be due to the cleavage of the protein by yeast protease. The purified protein was detected in SDS-PAGE as a smearing band in a range of 40-70 kDa (Fig. 1b) . The band pattern may be the result of glycosylation of multiple sites in the S1 protein by the yeast cells. The yield of the purified protein from one liter of yeast culture was found to be 180 μg.
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