Applied Microbiology and Biotechnology

, Volume 100, Issue 2, pp 661–671 | Cite as

Expression of a codon-optimised recombinant Ara h 2.02 peanut allergen in Escherichia coli

Biotechnologically relevant enzymes and proteins
First Online:
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Revised:
Accepted:

Abstract

Current diagnostic tools for peanut allergy using crude peanut extract showed low predictive value and reduced specificity for detection of peanut allergen-specific immunoglobulin E (IgE). The Ara h 2.02, an isoform of the major peanut allergen Ara h 2, contains three IgE epitope recognition sequence of ‘DPYSPS’ and may be a better reagent for component resolve diagnosis. This research aimed to generate a codon-optimised Ara h 2.02 gene for heterologous expression in Escherichia coli and allergenicity study of this recombinant protein. The codon-optimised gene was generated by PCR using overlapping primers and cloned into the pET-28a (+) expression vector. Moderate expression of a 22.5 kDa 6xhistidine-tagged recombinant Ara h 2.02 protein (6xHis-rAra h 2.02) in BL21 (DE3) host cells was observed upon induction with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). The insoluble recombinant protein was purified under denaturing condition using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography and refolded by dialysis in decreasing urea concentration, amounting to a yield of 74 mg/l of expression culture. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) and immunoblot analysis confirmed the production of the recombinant 6xHis-rAra h 2.02. The refolded recombinant 6xHis-rAra h 2.02, with or without adjuvant, was able to elicit comparable level of allergen-specific IgE and IgG1 in sensitised Balb/c mice. In addition, the specific IgE antibodies raised against the recombinant protein were able to recognise the native Ara h 2 protein, demonstrating its allergenicity and potential as a reagent for diagnosis and therapeutic study.

Keywords

Peanut allergen Ara h 2.02 Codon optimise pET-28a (+) vector Escherichia coli 
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Notes

Acknowledgments

This research was funded by the Research Grant Scheme (Proj-In-FAS-010) from Centre of Excellence for Research, Value Innovation and Entrepreneurship (CERVIE), UCSI University, Malaysia.

Compliance with ethical standards

ᅟ

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

The ethics for research involving animals was approved by the ethics committee of the Faculty of Applied Sciences, UCSI University.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.Faculty of Applied SciencesUCSI UniversityCherasMalaysia

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