Histrap hp ni 2 sepharose high performance

The allergen rPoly p 5 was acquired, as demonstrated by Bazon et al. [1 (link)]. Briefly, the rPoly p 5 cDNA was cloned in the pPICZαA vector and expressed in Komagataella phaffii (Pichia pastoris) X-33 cells. The soluble rPoly p 5 samples were subjected to purification by application in a prepacked column HisTrap HP™ (Ni⁺² Sepharose™ High Performance; GE Healthcare, Danderyd, Sweden), according to the manufacturer’s instructions. Afterwards, SDS-PAGE electrophoresis tests (12%) were performed to assess the purification process effectiveness. The purified protein was resuspended in phosphate-buffered saline (PBS, pH 7.4) for cell culture experiments.

The venom extracts and rPoly p 1 were obtained expressing the PLA1 cDNA cloned in specific vectors and expressed in E. coli-competent cells using the previously described protocols [12 (link),15 (link)]. For the rPoly p1 purification, the soluble fractions were applied to a prepacked column HisTrap HP™ (Ni⁺² Sepharose™ High Performance; GE Healthcare, Sweden) as described before [12 (link)], followed by SDS-PAGE (15%) analysis for monitoring the efficiency of the purification process.

The recombinant allergens rPoly p 1 and rPoly p 5 were obtained as described by Perez-Riverol et al. (2016) [29 (link)] and Bazon et al. (2017) [30 (link)], respectively. Briefly, the rPoly p 1 was obtained expressing the PLA1 cDNA cloned in PET-28a vector and expressed in E. coli BL21 (DE3) cells using the previously described protocol [29 (link)]. The rPoly p 5 were obtained, expressing the Ag5 cDNA cloned in pPICZαA vector and expressed in P. pastoris X-33 cells, using the previously described protocol [30 (link)]. For the rPoly p 1 and rPoly p 5 purification, the soluble fractions were applied to a prepacked column HisTrap HP™ (Ni⁺² Sepharose™ High Performance; GE Healthcare, Danderyd, Sweden), according to the manufacturer’s instructions, followed by SDS-PAGE (15%) analysis for monitoring the efficiency of the purification process.