Bacterial expression and purification of the amyloidogenic peptide PAPf39 for multidimensional NMR spectroscopy
Bacterial expression and purification of the amyloidogenic peptide PAPf39 for multidimensional NMR spectroscopy
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DOI：
10.1016/j.pep.2013.01.003
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发表时间：
2013-04-01
影响因子：
1.6
通讯作者：
Makhatadze, George I.
中科院分区：
文献类型：
--
作者：
Shanmuganathan, Aranganathan;Bishop, Anthony C.;Makhatadze, George I.
PAPf39是人前列腺酸性磷酸酶的39个残基多肽片段，在精液中形成淀粉样纤维。这些纤维被认为促进了艾滋病毒的传播。为了能够通过核磁共振光谱学研究PAPf39的结构，允许生产毫克量的同位素标记的多肽的有效方法是必不可少的。在这里，我们报道了统一标记的C-13和N-15标记的PAPf39肽的高效表达和纯化，通过在N-末端融合到泛素和在C-末端表达内含素。这使得通过核磁共振光谱研究PAPf39单体构象系综成为可能。为此，我们在低pH条件下对PAPf39多肽进行了单体状态下的核磁共振化学位移归属。(C)2013 Elsevier Inc.保留所有权利。
PAPf39 is a 39 residue peptide fragment from human prostatic acidic phosphatase that forms amyloid fibrils in semen. These fibrils have been implicated in facilitating HIV transmission. To enable structural studies of PAPf39 by NMR spectroscopy, efficient methods allowing the production of milligram quantities of isotopically labeled peptide are essential. Here, we report the high-yield expression and purification of uniformly C-13- and N-15-labeled PAPf39 peptide, through expression as a fusion to ubiquitin at the N-terminus and an intein at the C-terminus. This allows the study of the PAPf39 monomer conformational ensemble by NMR spectroscopy. To this end, we performed the NMR chemical shift assignment of the PAPf39 peptide in the monomeric state at low pH. (C) 2013 Elsevier Inc. All rights reserved.