Matchout deuterium labelling of proteins for small-angle neutron scattering studies using prokaryotic and eukaryotic expression systems and high cell-density cultures.
Matchout deuterium labelling of proteins for small-angle neutron scattering studies using prokaryotic and eukaryotic expression systems and high cell-density cultures.
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DOI：
10.1007/s00249-016-1186-2
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发表时间：
2017-07
期刊：
影响因子：
--
通讯作者：
Forsyth VT
中科院分区：
文献类型：
--
作者：
Dunne O;Weidenhaupt M;Callow P;Martel A;Moulin M;Perkins SJ;Haertlein M;Forsyth VT
小角中子散射（SANS）是表征大分子结构和相互作用的有力技术。其相对于其他溶液状态方法的主要优点是能够使用D2 O/H2O溶剂对比度变化来选择性地匹配多组分系统的特定部分。虽然蛋白质、核酸和脂质很容易以这种方式区分，但如果不通过选择性氘化引入额外的对比度，就不可能定位蛋白质-蛋白质系统的不同部分。在这里，我们描述了新的方法，通过该方法可以使用大肠杆菌和巴斯德毕赤酵母表达系统在高细胞密度培养物中产生“匹配标记”蛋白。该方法被设计为产生具有非常接近100%D2O的散射长度密度的蛋白质，当与复合物中的氢化伴侣蛋白质一起使用时提供清晰的对比。这允许产生单个样品系统，其中SANS测量在不同的溶剂对比度可以用于区分和建模的氢化组分，氘代组分，和整个复杂的。该方法具有显着的成本优势，已被广泛测试的两种类型的表达系统。
Small-angle neutron scattering (SANS) is a powerful technique for the characterisation of macromolecular structures and interactions. Its main advantage over other solution state approaches is the ability to use D2O/H2O solvent contrast variation to selectively match out specific parts of a multi-component system. While proteins, nucleic acids, and lipids are readily distinguished in this way, it is not possible to locate different parts of a protein–protein system without the introduction of additional contrast by selective deuteration. Here, we describe new methods by which ‘matchout labelled’ proteins can be produced using Escherichia coli and Pichia pastoris expression systems in high cell-density cultures. The method is designed to produce protein that has a scattering length density that is very close to that of 100% D2O, providing clear contrast when used with hydrogenated partner proteins in a complex. This allows the production of a single sample system for which SANS measurements at different solvent contrasts can be used to distinguish and model the hydrogenated component, the deuterated component, and the whole complex. The approach, which has significant cost advantages, has been extensively tested for both types of expression system.
影响因子：
5.6
作者：
Callow, P.;Sukhodub, A.;Taylor, James E. N.;Kneale, G. G.
通讯作者：
Kneale, G. G.