Recombinant Poly p Allergen Production

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.

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Abram D.M., Fernandes L.G., Perez-Riverol A., Regina Brochetto-Braga M, & Zollner R.D. (2020). Cross-Reactive Carbohydrate Determinant in Apis mellifera, Solenopsis invicta and Polybia paulista Venoms: Identification of Allergic Sensitization and Cross-Reactivity. Toxins, 12(10), 649.

Publication 2020

HisTrap HP™ (Ni+2 Sepharose™ High Performance;

Allergens Cdna Cells E coli Pla1 Sds page Sepharose Vector

Corresponding Organization : Universidade Estadual de Campinas (UNICAMP)

Other organizations : Universidade Estadual Paulista (Unesp)

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Variable analysis

independent variables

Expression of the PLA1 cDNA in pET-28a vector in E. coli BL21 (DE3) cells
Expression of the Ag5 cDNA in pPICZαA vector in P. pastoris X-33 cells

dependent variables

Purification of the recombinant allergens rPoly p 1 and rPoly p 5 using a prepacked column HisTrap HP™ (Ni+2 Sepharose™ High Performance; GE Healthcare, Danderyd, Sweden)
SDS-PAGE (15%) analysis for monitoring the efficiency of the purification process

control variables

Manufacturer's instructions for the prepacked column HisTrap HP™ (Ni+2 Sepharose™ High Performance; GE Healthcare, Danderyd, Sweden)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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