Ara h 2.02 protein as candidate protein for a better diagnosis Kelvin Lew Min Han and Renee Lim Lay Hong* Department of Biotechnology, Faculty of Applied Sciences, UCSI University, No. 1, Jalan Menara Gading, UCSI Heights, 56000 Cheras K.L., Malaysia. *Corresponding author:
[email protected]
INTRODUCTION
RESULTS AND DISCUSSION Recombinant Ara h 2.02-pET28a (+) construct
Recombinant Ara h 2.02 homology model
Peanut allergy is one of the most severe and fatal food hypersensitivity conditions. It is more common in western compared to Asian countries.1 About 0.4-0.6% children are affected by peanut allergy in Singapore and Philippine.4 However, the prevalence in Malaysia is still unknown. Currently there is no known cure for peanut allergy and prevention is by avoidance of peanuts.
(Kb)
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(bp)
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Additional IgE binding sites 6x His tag
Ara h 2 is a major peanut allergen identified, with reactivity to IgE in more than 90% of peanut-allergic patients.2
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600 500
PCR Product
The His tagged rAra h 2.02 structural homology model constructed based on X-ray rAra h 2.01-MBP (SWISS-MODEL and PyMoL analysis tool).5
Arah 2.02 gene (528bps) was cloned into the NheI and HindIII site of pET-28a vector. Positive clone was confirmed by PCR screening and DNA sequencing.
The rAra h 2.02 expressed as an insoluble protein
Overexpression of rAra h 2.02 induced in 1mM IPTG
Lane:
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Lane: M
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M
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(kDa)
Two isoforms exist, the Ara h 2.02 has an additional IgE epitope and higher IgE binding capacity.3 This more potent allergen for component resolved diagnosis of peanut allergy.
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The rAra h 2.02 protein (22.5kDa) was expressed in the insoluble fraction (I) after induction in 1mM IPTG at 4hr. No protein was found in the soluble fraction (S).
OBJECTIVES To produce the recombinant Ara h 2.02 protein in a bacteria expression system for characterisation.
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Increasing expression of rAra h 2.02 protein (22.5kDa) by Ara h 2.02/pET28a in BL21 cells was observed at time 1 to 4hr after 1mM IPTG induction. Expression of the His-tagged protein was confirmed on a Western immunoblot assay using HisDetector™
Purification in denaturing condition of 8M urea Lane: M
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Refolded rAra h 2.02 protein in PBS with 30% glycerol
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Lane: M
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METHODOLOGY In silico analysis of His-tagged rAra h 2.02 protein
(kDa)
Cloning of Ara h 2.02 gene into pET-28a (+) expression system
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Soluble His-tagged rAra h 2.02 protein (R) was obtained from refolding of the purified denatured protein (D). Yield of the refolded protein is 5.6mg/L of expression culture. C indicates the crude lysate from 4 hr expression culture.
CONCLUSION
Ni-NTA purification under 8M urea denaturing condition
The expression and purification of a soluble rAra h 2.02 protein is important for subsequent in vivo allergenicity test in Balbc/J mice and in development of a mouse model for therapeutic studies.
In vitro refolding by dialysing against decreasing urea concentration (6>4>2>1M) and 30% glycerol
REFERENCES
ACKNOWLEDGEMENT
1. Burks, A.W., Sampson, H.A., and Bannon, G.A. Review Article Series II. (1998) 53:725–730. 2. 3.
Balb/c mice immunisation and allergenicity testing
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The His-tagged rAra h 2.02 protein (22.5kDa) was purified using Ni-NTA affinity column. It was eluted at pH 5.3 in 8M urea (F1-F7). The rAra h 2.02 protein was confirmed by MALDI-TOF analysis.
Small scale expression study and solubility test of rArah 2.02 protein
MALDI-TOF mass spectrometry and immunoblot analysis
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4. 5.
Chatel, J.M., Bernard, H., Orson, F.M., Journal of International Archieve Allergy Immunology (2003) ;131:14-18 Lehmann, K., Hoffmann, S., Neudecker, P., et al. Protein Expression and Purification (2003) 31, 250–259 Shrek, L.P.C., Cabrera-Morales, E.A., Soh, S.E., et al. Journal of American Academy of Allergy, Asthma & Immunology (2010) 324-331. Mueller, G., Gosavi, R., & Pomés, A., et al. Allergy (2010), 66(7), 878–885.
This research is supported by the RGS grant Proj-In-FAS-010 from UCSI University.
