The sandfly salivary protein used during the optimization process of the vaccine candidate (his-tagged) was produced using a mammalian expression system, as explained elsewhere [20 (link)]. LJL143 used in the antigenicity pre-clinical assay per-se (non his-tagged) was obtained using a yeast expression system (more cost-effective). Briefly, DNA coding for LJL143 without the signal peptide was codon optimized based on Pichia pastoris usage preference and subcloned into Pichia secretory expression vector pPICZαA (Invitrogen) using EcoRI/XbaI restriction sites. The correct insert sequence and reading frame of recombinant plasmid was confirmed by double-stranded sequencing using vector flanking primers α-factor and 3’AOX-1 and then transformed into Pichia pastoris X-33 by electroporation. The expression of LJL143 was induced with 0.5% methanol at 30°C for 72 hours and the highest expression clone was chosen for making seed stock with 20% glycerol. Large-scale expression of LJL143 was induced with methanol in 10L fermentation. Coomassie G-250 (Simply Blue) stained NuPAGE Bis-Tris gels (Invitrogen) were used to assess the purity of the recombinant protein.
Pichia secretory expression vector ppiczαa
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pichia secretory expression vector pPICZαA is a tool used for the expression and secretion of recombinant proteins in the yeast Pichia pastoris. It contains the AOX1 promoter for methanol-inducible expression, the Saccharomyces cerevisiae α-factor secretion signal for protein secretion, and the Zeocin resistance gene for selection of transformed Pichia cells.
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3 protocols using pichia secretory expression vector ppiczαa
1
Production and Purification of LJL143 Protein
2
Recombinant SARS-CoV-2 Spike RBD Production
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The DNAs encoding RBD219-WT (residues 331–549 of the SARS-CoV-2 spike protein, GenBank: QHD43416.1), RBD219-N1 (residues 332–549), and RBD219-N1C1 (residues 332–549, C538A) were codon-optimized based on yeast codon usage preference and synthesized by GenScript (Piscataway, NJ, USA), followed by subcloning into the Pichia secretory expression vector pPICZαA (Invitrogen) using EcoRI/XbaI restriction sites. A hexahistidine tag version for RBD219-WT and RBD219-N1 (namely, RBD219-WT + His and RBD219-N1 + His, respectively) was also generated by adding additional DNA encoding six histidine residues at the C-terminus to facilitate purification as a backup. The recombinant plasmid DNAs were then transformed into Pichia pastoris X-33 by electroporation. The expression of the recombinant RBDs was confirmed by induction with 0.5% methanol at 30 °C for 72 h. The seed stock in 20% glycerol of each recombinant construct was then generated as described previously [13 (link)].
3
Recombinant SARS-CoV-2 Spike Protein Production
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