Production and Purification of LJL143 Protein

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.