β2-microglobulin can be refolded into a native state from ex vivo amyloid fibrils
β2-microglobulin can be refolded into a native state from ex vivo amyloid fibrils
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DOI：
10.1046/j.1432-1327.1998.2580061.x
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发表时间：
1998-11-15
期刊：
影响因子：
--
通讯作者：
Ferri, G
中科院分区：
文献类型：
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作者：
Bellotti, V;Stoppini, M;Ferri, G
β 2-微球蛋白原纤维已经从已经接受慢性血液透析11年的患者的股骨头中提取。年蛋白质N-末端部分的一级结构和电喷雾质谱法的质量测定表明，通过水提取程序提取为原纤维的β 2-微球蛋白未被糖化，Asn 17未被脱酰胺。在超过20%的β 2-微球蛋白分子中观察到有限的蛋白水解，主要裂解位点位于Lys 6和Tyr 10的C-末端。通过在6 M Gdn/HCl中的凝胶过滤纯化来自原纤维的β 2-微球蛋白，并进行重折叠程序。通过对天然蛋白去折叠途径的研究，确定了复性条件。β 2-微球蛋白在中性pH下是稳定的，在中性pH下它比在酸性条件下显示出更低的自聚集倾向。脉冲稀释和在pH 7.5的重折叠缓冲液中的广泛透析产生具有与天然形式相同的三级结构的β 2-微球蛋白。近紫外区的CD光谱和Trp的固有荧光光谱与天然蛋白重叠，但远紫外区的CD光谱受到β 2-微球蛋白片段产生的寡聚体的影响，这些片段降低了天然β 2-微球蛋白典型的205 nm处的正光偏振。
beta 2-microglobulin fibrils have been extracted from the femoral head of a patient who has been under chronic haemodialysis for 11. years. The primary structure of the N-terminal portion of the protein and mass determination by electrospray mass spectrometry demonstrate that beta 2-microglobulin, extracted as fibrils by the water extraction procedure, was not glycated and that Asn17 was not deamidated. Limited proteolysis was observed in more than 20% of beta 2-microglobulin molecules and the main cleavage sites were at the C-terminus of Lys6 and Tyr10. beta 2-microglobulin from fibrils has been purified by gel filtration in 6 M Gdn/HCl and submitted to a refolding procedure. The refolding conditions have been determined through the study of the unfolding pathway of the native protein. beta 2-microglobulin is stable at neutral pH where it displays a lower tendency to self-aggregate than in acidic conditions. Pulse dilution and extensive dialysis in refolding buffer at pH 7.5 yields beta 2-microglobulin with a tertiary structure identical to that of the native form. The CD spectrum in the near-ultraviolet region and the spectrum of the intrinsic fluorescence of Trp overlap those of the native protein, but the CD spectrum in the far-ultraviolet region is affected by the contribution of oligomers created by beta 2-microglobulin fragments that reduce the positive light polarisation at 205 nm typical of native beta 2-microglobulin.