Expression of a codon-optimised recombinant Ara h 2.02 peanut allergen in Escherichia coli
Two isoforms exist, namely Ara h 2.01 and Ara h 2.02. Ara h 2.02 (19 kDa) is 12 amino acids longer due to an additional IgE binding epitope (of the sequence ‘DPYSPS’) and higher IgE binding reactivity and is a more potent cross-linker of IgE compared to Ara h 2.01. Hence, the Ara h 2.02 isoform may be a more sensitive diagnostic reagent for peanut allergy.
The Ara h 2.01 in pET-16b vector was expressed as 90–95 % inclusion bodies; however, protein solubility was improved by 50 % using the pET-32a vector in Escherichia coli AD494 (DE3) and Origami (DE3) hosts, with a reported yield of 30–46 mg/ l culture.
The native Ara h 2.02 codon sequence was changed, yet retaining the same amino acid sequence to achieve, where possible, a closer percentage of codon frequen- cies to that of highly expressed E. coli genes
The codon-optimised Ara h 2.02 gene has higher CAI (N=0.55; S=0.64) and CFD value (N=39; S=45) com- pared to the native gene sequence, hence predicting a higher expression in E. coli host.
A 22.5 kDa protein band of the 6xHis-rAra h 2.02 was observed at 1 h after IPTG induction, which was absent in the uninduced sample (t=0) and the expression culture of pET-28a/BL21 (negative control).
The recombinant protein binding was optimal at pH 7.8 as less of the protein appears in the lysate flow-through from the resin (F1 and F20)
Eluted fractions of 6xHis-rAra h 2.02 purified in 8 M urea were pooled and refolded by stepwise dialysis in descending urea concentration, allowing for the gradual removal of urea.
The total yield of the denatured and refolded 6xHis-rAra h 2.02 protein from a 50 ml expression culture of p2/BL21 was 0.712 and 0.370 mg, respectively. Protein recovery was estimated to be only 52 % due to protein loss in the dialysis and refolding process. The estimated yield of the refolded 6xHis-rAra h 2.02 was 74 mg/l of the expression culture.
The 6xHis-rAra h 2.02-specific IgG1 and IgE were detected on the second week (day 14), after the first subcutaneous administration of mice in groups A and B, but were absent throughout for the control (PBS) mice group. Mice in both groups A and B showed detectable specific IgG2a at day 14 that increases with subsequent administration, despite the low level.
The native Ara h 2 showed higher specific reactivity to IgE from mice immunized by the recombinant 6xHis-rAra h 2.02 protein.
Imidazole and glycerol in the binding and wash buffer helped to prevent nonspecific protein binding, therefore increasing efficiency of the Ni-NTA affinity purification. Washing performed at decreasing pH (7.8, 6.5 and 6.3) was able to remove substantial E. coli proteins. 30 % glycerol was added during refolding by dialysis to prevent protein aggregation.
Allergen-specific IgE and IgG1 are progressively produced during sensitisation and subsequent exposures to allergen. IgG1 plays a significant role in Th2 response resulting in the classic IgE-mediated and alternative FcγRIII-mediated murine response during anaphylactic reaction upon challenge.
The IgG2a antibodies, on the contrary, are responsible for delayed type hypersensitivity (DTH) caused by Th1 responses in mice where IgG2a increases with attenuation of IgE and and IgG1.