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Purification and characterization of an amyloidogenic repeat domain from the functional amyloid Pmel17
Protein Expression and Purification ( IF 1.4 ) Pub Date : 2021-07-20 , DOI: 10.1016/j.pep.2021.105944
Dexter N Dean 1 , Jennifer C Lee 1
Affiliation  

The pre-melanosomal protein (Pmel17) is a human functional amyloid that supports melanin biosynthesis within melanocytes. This occurs in the melanosome, a membrane-bound organelle with an acidic intraluminal pH. The repeat region of Pmel17 (RPT, residues 315–444) has been previously shown to form amyloid aggregates under acidic melanosomal conditions, but not under neutral cytosolic conditions, when expressed and purified using a C-terminal hexa-histidine tag (RPT-His). Given the importance of protonation states in RPT-His aggregation, we questioned whether the histidine tag influenced the pH-dependent behavior. In this report, we generated a tagless RPT by inserting a tobacco etch virus (TEV) protease recognition sequence (ENLYGQ(G/S)) immediately upstream of a native glycine residue at position 312 in Pmel17. After purification of the fusion construct using a histidine tag, cleavage with TEV protease generated a fully native RPT (nRPT) spanning resides 312–444. We characterized the aggregation of nRPT, which formed amyloid fibrils under acidic conditions (pH ≤ 6) but not at neutral pH. Characterizing the morphologies of nRPT aggregates using transmission electron microscopy revealed a pH-dependent maturation from short, curved structures at pH 4 to paired, rod-like fibrils at pH 6. This was accompanied by a secondary structural transition from mixed random coil/β-sheet at pH 4 to canonical β-sheet at pH 6. We also show that pre-formed nRPT fibrils undergo disaggregation upon dilution into pH 7 buffer. More broadly, this strategy can be utilized to generate native amyloidogenic domains from larger proteins by utilizing intrinsic N-terminal glycine or serine residues.



中文翻译:

从功能性淀粉样蛋白 Pmel17 中纯化和表征淀粉样蛋白重复结构域

前黑素体蛋白 (Pmel17) 是一种人类功能性淀粉样蛋白,可支持黑素细胞内的黑色素生物合成。这发生在黑素体中,这是一种膜结合的细胞器,具有酸性的腔内 pH 值。Pmel17 的重复区域(RPT,残基 315-444)先前已显示在酸性黑素体条件下形成淀粉样蛋白聚集体,但在中性细胞溶质条件下,当使用 C 端六组氨酸标签(RPT-His)表达和纯化时)。鉴于质子化状态在 RPT-His 聚集中的重要性,我们质疑组氨酸标签是否影响 pH 依赖性行为。在本报告中,我们通过在 Pmel17 中 312 位的天然甘氨酸残基上游插入烟草蚀刻病毒 (TEV) 蛋白酶识别序列 (ENLYGQ(G/S)) 来生成无标签 RPT。在使用组氨酸标签纯化融合构建体后,用 TEV 蛋白酶切割产生了一个完全天然的 RPT (nRPT),跨越 312-444。我们表征了 nRPT 的聚集,它在酸性条件(pH ≤ 6)下形成淀粉样蛋白原纤维,但在中性 pH 下不形成。使用透射电子显微镜表征 nRPT 聚集体的形态揭示了 pH 依赖性成熟,从 pH 4 的短弯曲结构到 pH 6 的成对棒状原纤维。这伴随着从混合无规线圈/β-的二级结构转变在 pH 4 的片层到 pH 6 的典型 β-片层。我们还表明,预先形成的 nRPT 原纤维在稀释到 pH 7 缓冲液中时会发生解聚。更广泛地,

更新日期:2021-07-23
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