In vitro stability and immunoreactivity of the native and recombinant plant food 2S albumins Ber e 1 and SFA-8
Article first published online: 5 AUG 2003
Clinical & Experimental Allergy
Volume 33, Issue 8, pages 1147–1152, August 2003
How to Cite
Murtagh, G. J., Archer, D. B., Dumoulin, M., Ridout, S., Matthews, S., Arshad, S. H. and Alcocer, M. J. C. (2003), In vitro stability and immunoreactivity of the native and recombinant plant food 2S albumins Ber e 1 and SFA-8. Clinical & Experimental Allergy, 33: 1147–1152. doi: 10.1046/j.1365-2222.2003.01736.x
- Issue published online: 5 AUG 2003
- Article first published online: 5 AUG 2003
- Submitted 28 November 2002; revised 19 February 2003; accepted 13 May 2003
- Brazil nut;
- recombinant allergens;
- 2S albumins;
Background The ability of an intact protein to reach the circulatory system may be a prerequisite to allergenicity and many allergens, particularly those from plant foods, have been found to be consistently more resistant to digestion by pepsin than other proteins.
Objective This study assessed the pepsinolytic stability of native 2S albumins from Brazil nut and sunflower seed and their recombinant versions produced in Pichia pastoris. The physicochemical stability of native and recombinant Brazil nut 2S albumins and recombinant sunflower seed 2S albumin was also assessed. The immunoreactivity of native Brazil nut 2S albumin and recombinant 2S albumins was compared using serum from patients allergic to Brazil nuts and animals immunized with native 2S albumins.
Methods Digestibility was measured in simulated gastric fluid followed by SDS-PAGE. Circular dichroism spectra were used to analyse unfolding, as proteins were denatured by temperature, pH and guanidinium chloride. Immunoreactivity was assessed by immunoblot, RAST and ELISA.
Results Brazil nut 2S albumin was significantly more resistant to proteolytic digestion than other Brazil nut proteins. It was also resistant to thermally and chemically induced denaturation. Equally high resistance to proteolytic digestion was observed with sunflower seed 2S albumin. The recombinant albumins mirrored their native counterparts in stability and immunoreactivity.
Conclusion The important food allergen Brazil nut 2S albumin is as stable to digestion as is sunflower seed 2S albumin, whose allergenicity has yet to be determined. The 2S albumins and their recombinant counterparts could not be easily denatured by physicochemical treatments. The results suggest that 2S albumin is the only Brazil nut protein to reach the gut immune system intact. The production of properly folded recombinant proteins will facilitate mechanistic studies as well as diagnostic testing and antigen-based therapies.