Long Fei, Cho Wonhwa, Ishii Yoshitaka
Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, United States.
Protein Expr Purif. 2011 Sep;79(1):16-24. doi: 10.1016/j.pep.2011.05.012. Epub 2011 May 27.
Amyloid fibrils of Alzheimer's β-amyloid peptide (Aβ) are a primary component of amyloid plaques, a hallmark of Alzheimer's disease (AD). Enormous attention has been given to the structural features and functions of Aβ in amyloid fibrils and other type of aggregates in associated with development of AD. This report describes an efficient protocol to express and purify high-quality 40-residue Aβ(1-40), the most abundant Aβ in brains, for structural studies by NMR spectroscopy. Over-expression of Aβ(1-40) with glutathione S-transferase (GST) tag connected by a Factor Xa recognition site (IEGR(▾)) in Escherichia coli resulted in the formation of insoluble inclusion bodies even with the soluble GST tag. This problem was resolved by efficient recovery of the GST-Aβ fusion protein from the inclusion bodies using 0.5% (w/v) sodium lauroyl sarcosinate as solubilizing agent and subsequent purification by affinity chromatography using a glutathione agarose column. The removal of the GST tag by Factor Xa enzymatic cleavage and purification by HPLC yielded as much as ∼7 mg and ∼1.5mg of unlabeled Aβ(1-40) and uniformly (15)N- and/or (13)C-protein Aβ(1-40) from 1L of the cell culture, respectively. Mass spectroscopy of unlabeled and labeled Aβ and (1)H/(15)N HSQC solution NMR spectrum of the obtained (15)N-labeled Aβ in the monomeric form confirmed the expression of native Aβ(1-40). It was also confirmed by electron micrography and solid-state NMR analysis that the purified Aβ(1-40) self-assembles into β-sheet rich amyloid fibrils. To the best of our knowledge, our protocol offers the highest yields among published protocols for production of recombinant Aβ(1-40) samples that are amendable for an NMR-based structural analysis. The protocol may be applied to efficient preparation of other amyloid-forming proteins and peptides that are (13)C- and (15)N-labeled for NMR experiments.
阿尔茨海默病β-淀粉样肽（Aβ）的淀粉样纤维是淀粉样斑块的主要成分，而淀粉样斑块是阿尔茨海默病（AD）的一个标志。人们对Aβ在淀粉样纤维及与AD发展相关的其他类型聚集体中的结构特征和功能给予了极大关注。本报告描述了一种高效的方案，用于表达和纯化高质量的40个残基的Aβ(1-40)，这是大脑中最丰富的Aβ，用于通过核磁共振光谱进行结构研究。在大肠杆菌中，通过一个凝血因子Xa识别位点（IEGR(▾)）连接谷胱甘肽S-转移酶（GST）标签来过量表达Aβ(1-40)，即使带有可溶的GST标签，也会导致形成不溶性包涵体。使用0.5%（w/v）月桂酰肌氨酸钠作为增溶剂从包涵体中高效回收GST-Aβ融合蛋白，并随后使用谷胱甘肽琼脂糖柱通过亲和色谱进行纯化，解决了这个问题。通过凝血因子Xa酶切去除GST标签并通过高效液相色谱（HPLC）进行纯化，从1升细胞培养物中分别获得了多达约7毫克和约1.5毫克的未标记Aβ(1-40)以及均匀（15）N-和/或（13）C-蛋白Aβ(1-40)。未标记和标记的Aβ的质谱以及所获得的以单体形式存在的（15）N标记的Aβ的（1）H/（15）N HSQC溶液核磁共振谱证实了天然Aβ(（1-40）的表达。通过电子显微镜和固态核磁共振分析也证实，纯化的Aβ(1-40)会自组装成富含β-折叠的淀粉样纤维。据我们所知，在已发表的用于生产适用于基于核磁共振的结构分析的重组Aβ(1-40)样品的方案中，我们的方案产量最高。该方案可应用于高效制备其他用于核磁共振实验的、经（13）C-和（15）N-标记的淀粉样形成蛋白和肽。
