Murtagh G J, Archer D B, Dumoulin M, Ridout S, Matthews S, Arshad S H, Alcocer M J C
School of Life and Environmental Sciences, University of Nottingham, University Park, Nottingham, UK.
Clin Exp Allergy. 2003 Aug;33(8):1147-52. doi: 10.1046/j.1365-2222.2003.01736.x.
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.
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.
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.
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.
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.
完整蛋白质进入循环系统的能力可能是引发过敏的一个先决条件，并且已发现许多过敏原，尤其是来自植物性食物的过敏原，相较于其他蛋白质，对胃蛋白酶消化的耐受性更强。
本研究评估了来自巴西坚果和向日葵籽的天然2S白蛋白及其在毕赤酵母中产生的重组形式对胃蛋白酶水解的稳定性。还评估了天然和重组巴西坚果2S白蛋白以及重组向日葵籽2S白蛋白的物理化学稳定性。使用对巴西坚果过敏患者的血清以及用天然2S白蛋白免疫的动物血清，比较了天然巴西坚果2S白蛋白和重组2S白蛋白的免疫反应性。
在模拟胃液中测量消化率，随后进行SDS-PAGE。由于蛋白质会因温度、pH值和氯化胍而变性，因此使用圆二色光谱分析其解折叠情况。通过免疫印迹、RAST和ELISA评估免疫反应性。
巴西坚果2S白蛋白比其他巴西坚果蛋白质对蛋白水解消化的耐受性明显更强。它也耐受热诱导和化学诱导的变性。向日葵籽2S白蛋白对蛋白水解消化也具有同样高的耐受性。重组白蛋白在稳定性和免疫反应性方面与其天然对应物相似。
重要的食物过敏原巴西坚果2S白蛋白与向日葵籽2S白蛋白（其致敏性尚未确定）在消化稳定性方面相当。2S白蛋白及其重组对应物不易被物理化学处理变性。结果表明，2S白蛋白是唯一能完整到达肠道免疫系统的巴西坚果蛋白质。正确折叠的重组蛋白的产生将有助于进行机制研究以及诊断测试和基于抗原的治疗。
