用于多维核磁共振波谱分析的淀粉样蛋白生成肽PAPf39的细菌表达与纯化。
Bacterial expression and purification of the amyloidogenic peptide PAPf39 for multidimensional NMR spectroscopy.
作者信息
Shanmuganathan Aranganathan, Bishop Anthony C, French Kinsley C, McCallum Scott A, Makhatadze George I
机构信息
Center for Biotechnology and Interdisciplinary Studies and Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
出版信息
Protein Expr Purif. 2013 Apr;88(2):196-200. doi: 10.1016/j.pep.2013.01.003. Epub 2013 Jan 11.
Abstract
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 (13)C- and (15)N-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.
摘要
PAPf39是一种来自人前列腺酸性磷酸酶的39个残基的肽片段，它在精液中形成淀粉样纤维。这些纤维被认为有助于HIV传播。为了通过核磁共振光谱对PAPf39进行结构研究，高效生产毫克级同位素标记肽的方法至关重要。在此，我们报告了通过在N端与泛素融合以及在C端与内含肽融合表达，实现了均匀（13）C和（15）N标记的PAPf39肽的高产表达和纯化。这使得能够通过核磁共振光谱研究PAPf39单体的构象集合。为此，我们在低pH下对单体状态的PAPf39肽进行了核磁共振化学位移归属。
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