Urea-assisted reconstitution of discoidal high-density lipoprotein
Urea-assisted reconstitution of discoidal high-density lipoprotein
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尿素辅助盘状高密度脂蛋白重建
DOI：
10.1021/acs.biochem.0c00075
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发表时间：
2020
期刊：
影响因子：
2.9
通讯作者：
T.
中科院分区：
文献类型：
--
作者：
Fukuda;R.; Saito;M.; Shibukawa;S.; Sumino;A.; Nakano;M.; Murakami;T.
高密度脂蛋白（HDL）是一种天然存在的脂质和脂质结合蛋白的复合物。胆酸盐透析法由Jonas于1969年首次报道，是在试管中用磷脂和最主要的蛋白质载脂蛋白A-1（apoA-I）重建盘状HDL（dHDL）的最广泛使用的方法。在这里，我们表明，dHDL相关的复合物也可以通过轻轻地混合1，2-二肉豆蔻酰-sn-甘油-3-磷酸胆碱（DMPC）和apoA-I或其突变体在乙醇/水溶液中含有尿素在几摩尔浓度，然后孵育的混合物在凝胶液晶相转变温度在试管中。随后的纯化步骤包括尺寸排阻色谱法后的快速透析。所得HDL样纳米颗粒（命名为uHDL）的产率（分别为73 ± 3%和70 ± 1%蛋白质和DMPC）与胆酸盐透析方法中获得的68 ± 9%和71 ± 12%的值相当。利用apoA-I和两个突变体，发现该方法中的关键因素是在折叠和未折叠转变中点浓度的尿素。通过在疏水药物全反式维甲酸（ATRA）存在下使用这种尿素辅助的方法，也可以一步制备ATRA负载的uHDL。负载效率与通过胆酸盐透析法重构的ATRA和uHDL或dHDL的混合中的负载效率相当。原子力显微镜分析显示，uHDL和ATRA负载的uHDL是盘状的。我们的尿素辅助方法是一种简单有效的方法，用于重建dHDL，并可用于制备各种药物-dHDL复合物。
High-density lipoprotein (HDL) is a naturally occurring composite of lipids and lipid-binding proteins. The cholate dialysis method, first reported by Jonas in 1969, is the most widely used approach for reconstituting discoidal HDL (dHDL) in test tubes with phospholipids and the most dominant protein, apolipoprotein A-1 (apoA-I). Here, we show that a dHDL-relevant complex can also be prepared by gently mixing 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and apoA-I or its mutants in ethanol/H2O solutions containing urea at a concentration of a few molar and then incubating the mixture at the gel–liquid crystalline phase transition temperature in test tubes. Subsequent purification steps involve quick dialysis following size exclusion chromatography. The yields (73 ± 3% and 70 ± 1% protein and DMPC, respectively) of the resulting HDL-like nanoparticles, designated as uHDL, were comparable to the values of 68 ± 9% and 71 ± 12% obtained in the cholate dialysis method. Using apoA-I and two mutants, the key factor in this method was found to be urea at the folded and unfolded transition midpoint concentration. By using this urea-assisted method in the presence of a hydrophobic drug, all-trans-retinoic acid (ATRA), one-step preparation of ATRA-loaded uHDL was also possible. The loading efficiency was comparable to that in the mixing of ATRA and uHDL or dHDL reconstituted by the cholate dialysis method. Atomic force microscopy analysis revealed that uHDL and ATRA-loaded uHDL were discoidal. Our urea-assisted method is an easy and efficient method for reconstituting dHDL and can be utilized to prepare various drug–dHDL complexes.