Supplementary Material for: A novel quality control strategy for the preparation of high-quality recombinant allergens in Escherichia coli: a case study of Der f 2

Introduction
Recombinant allergens produced by Escherichia coli (E. coli) system play an important role in the component-resolved diagnostics of allergy and vaccine development. However, incorrect folding of recombinant allergens may affect their application. Therefore, it is very important to monitor the correct folding of recombinant allergens. Currently, there is still a lack of a quality control strategy to solve this problem. In this study, a mite allergen, Der f 2, was taken as an example to establish a novel quality control strategy, which was based on chromatography to isolate the allergen, and on enzyme-linked immunosorbent assay to verify the IgE reaction of the isolated allergen.
Methods
The nucleotide sequence encoding Der f 2 was codon-optimized and cloned into pET-28a (+) plasmid. Best conditions for Der f 2 expression in E. coli were sought. The inclusion body of Der f 2 was denatured and purified by nickel affinity chromatography. Refolding processes were compared using glutathione redox system. The fully folded and partially folded proteins were separated by anion exchange chromatography, and the IgE reactivity of the isolated proteins was verified by indirect enzyme-linked immunosorbent assay.
Results
An optimized 387 bp segment of the Der f 2 coding gene was successfully expressed in E. coli. Best induction conditions included pre-induction bacterial density with absorbance value at 600 nm was 0.6, 1 mM IPTG at 28 ℃ for 4 h. The Der f 2 protein after refolding was separated by chromatography and two fractions were obtained. The first fraction was identified as monomer protein and the second as aggregate by size-exclusion chromatography. Indirect enzyme-linked immunosorbent assay also confirmed that the first fraction showed higher IgE reactivity.
Conclusion
In this study, a novel quality control strategy based on chromatographic separation and IgE reactivity monitoring was established in the case of mite Der f 2, which systematically evaluated the effectiveness of multiple preparation methods for the first time. It is faster and more convenient, when compared with existing methods such as size-exclusion chromatography. This strategy laid a foundation for the stable application of recombinant allergens produced by E. coli in component-resolved diagnostics and the development of molecular vaccines in the future.