Solubilisation & purification of membrane proteins using benzylamine-modified SMA polymers

IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Biophysical chemistry Pub Date : 2024-10-18 DOI:10.1016/j.bpc.2024.107343
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Abstract

Extraction of proteins from the membrane using styrene maleic acid co-polymers (SMA), forming SMA lipid particles (SMALPs), has allowed for the first time the purification of membrane proteins with their lipid bilayer environment. To date, SMA2000 has been the most effective polymer used for this purpose, with a 2:1 ratio of styrene:maleic acid, and styrene and maleic acid moieties spread statistically throughout the chain. However, SMA2000 is a highly polydisperse polymer that contains an array of different polymer lengths and sequences. RAFT polymerisation offers much better control over the polymer length; however, homogeneous distribution of styrene and maleic acid throughout the polymer is difficult to achieve. Instead, here RAFT polymerisation was used to produce a 1:1 styrene:maleic anhydride polymer, which was then modified with benzylamine. This mimics the 2:1 hydrophobic:hydrophilic nature of SMA2000, while controlling the length and obtaining a homogeneous distribution of the hydrophobic moieties (styrene and N-benzylmaleimide). SMA-benzylamine (SMA-BA) polymers of three different lengths (2, 4, and 7 kDa) were all able to solubilise purified lipids, cellular membranes, and a range of specific proteins. However, the larger 7 kDa polymer solubilised membranes more slowly and less efficiently than the shorter polymers. This also affected the yield of purified protein obtained by affinity purification with this polymer. The smallest 2 kDa polymer solubilised membranes the fastest but appeared to offer less stability to the extracted proteins. The SMA-BA polymers were more sensitive to Mg2+ ions than SMA2000. SMA-BA 4 kDa was otherwise comparable to SMA2000 and even gave a higher degree of purity.

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使用苄胺改性 SMA 聚合物增溶和纯化膜蛋白。
使用苯乙烯-马来酸共聚物(SMA)从膜中提取蛋白质,形成 SMA 脂质颗粒(SMALPs),首次实现了在脂质双分子层环境中纯化膜蛋白质。迄今为止,SMA2000 是用于此目的最有效的聚合物,其苯乙烯和马来酸的比例为 2:1,苯乙烯和马来酸分子分布在整个链中。不过,SMA2000 是一种高度分散的聚合物,含有一系列不同长度和序列的聚合物。RAFT 聚合可以更好地控制聚合物的长度;但是,很难实现苯乙烯和马来酸在整个聚合物中的均匀分布。因此,这里采用了 RAFT 聚合法来生产 1:1 苯乙烯:马来酸酐聚合物,然后用苄胺对其进行改性。这就模仿了 SMA2000 2:1 的疏水:亲水性质,同时控制了长度并获得了疏水分子(苯乙烯和 N-苄基马来酰亚胺)的均匀分布。三种不同长度(2、4 和 7 kDa)的 SMA-苄胺(SMA-BA)聚合物都能溶解纯化的脂质、细胞膜和一系列特定蛋白质。不过,与较短的聚合物相比,较大的 7 kDa 聚合物溶解膜的速度更慢,效率更低。这也影响了使用这种聚合物进行亲和纯化所获得的纯化蛋白质的产量。最小的 2 kDa 聚合物溶解膜的速度最快,但提取蛋白质的稳定性似乎较差。与 SMA2000 相比,SMA-BA 聚合物对 Mg2+ 离子更敏感。SMA-BA 4 kDa 在其他方面与 SMA2000 相当,甚至纯度更高。
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来源期刊
Biophysical chemistry
Biophysical chemistry 生物-生化与分子生物学
CiteScore
6.10
自引率
10.50%
发文量
121
审稿时长
20 days
期刊介绍: Biophysical Chemistry publishes original work and reviews in the areas of chemistry and physics directly impacting biological phenomena. Quantitative analysis of the properties of biological macromolecules, biologically active molecules, macromolecular assemblies and cell components in terms of kinetics, thermodynamics, spatio-temporal organization, NMR and X-ray structural biology, as well as single-molecule detection represent a major focus of the journal. Theoretical and computational treatments of biomacromolecular systems, macromolecular interactions, regulatory control and systems biology are also of interest to the journal.
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