Recovery and purification of highly aggregation-prone disulfide-containing peptides: Application to islet amyloid polypeptide
Recovery and purification of highly aggregation-prone disulfide-containing peptides: Application to islet amyloid polypeptide
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DOI：
10.1016/j.ab.2005.11.029
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发表时间：
2006-04-15
影响因子：
2.9
通讯作者：
Raleigh, DP
中科院分区：
文献类型：
--
作者：
Abedini, A;Singh, G;Raleigh, DP
胰岛淀粉样多肽（IAPP）是一种由37个氨基酸残基组成的胰腺激素。它负责体内胰岛淀粉样蛋白的形成，并且在体外非常不溶且易于聚集，特别是在碱性pH下。该肽在残基2和7之间含有二硫键和酰胺化C末端。没有报道用于生产酰胺化IAPP的表达系统。该肽难以合成，并且通过传统方法形成二硫键是有问题的。我们已经发现，使用1，1，1，3，3，3-六氟-2-丙醇（HFIP）或二甲亚砜（DMSO）显著改善了二硫化物形成和高度聚集倾向IAPP序列的纯化。这些有机溶剂的使用增加了疏水性肽的溶解度，避免了碱性水溶液的使用，并且消除了在氧化形成Cys-2至Cys-7二硫桥期间连续搅拌的需要。消除搅拌步骤和碱性溶液有助于减少聚集，并允许更一致的高效液相色谱（HPLC）保留时间。发现使用DMSO形成分子内二硫化物是IAPP氧化的最有效方法，将反应时间从24小时缩短至5小时。聚集的IAPP可以用HFIP或DMSO重新溶解，并通过HPLC以非常好的产率回收。(c)2005年爱思唯尔公司All rights reserved.
Islet amyloid polypeptide (IAPP) is a 37-residue pancreatic hormone. It is responsible for the formation of islet amyloid in vivo and is very insoluble and aggregation-prone in vitro, particularly at basic pH. The peptide contains a disulfide bridge between residues two and seven and an amidated C terminus. There is no reported expression system for the production of amidated IAPP. The peptide is difficult to synthesize and formation of the disulfide by traditional method is problematic. We have found that the use of I,l,l,3,3,3-hexafluoro-2-propanol (HFIP) or dimethyl sulfoxide (DMSO) significantly improves disulfide formation and purification of highly aggregation-prone IAPP sequences. The use of these organic solvents increases the Solubility of the hydrophobic peptides, avoids the use of aqueous basic solutions, and eliminates the need for continuous stirring during oxidation to form the Cys-2 to Cys-7 disulfide bridge. Elimination of the stirring step and basic Solution helps to reduce aggregation and allows for more consistent high-performance liquid chromatography (HPLC) retention times. Formation of the intramolecular disulfide using DMSO was found to be the most effective method for IAPP oxidation, reducing the reaction time from 24 to 5 h. Aggregated IAPP can be resolubilized by HFIP or DMSO and recovered by HPLC with very good yield. (c) 2005 Elsevier Inc. All rights reserved.