Christiansen K, Carlsen J
Biochim Biophys Acta. 1983 Nov 9;735(2):225-33. doi: 10.1016/0005-2736(83)90297-3.
A method is described for reconstitution of a protein into lipid vesicles using one of the natural detergents lysophosphatidylcholine or lysophosphatidic acid. The intestinal microvillus enzyme, aminopeptidase N (EC 3.4.11.2) is incorporated into lipid vesicles prepared from a total lipid extract of the microvillus membrane. The method is based on fusion of aminopeptidase-lysophospholipid micelles with liposomes prepared by sonication. The incorporation of the protein into the lipid bilayer is analyzed by gel permeation chromatography and sucrose density gradient centrifugation. The coincidence of the protein and lipid profiles is used to evaluate protein incorporation. The incorporation is visualized by electron microscopy with negative staining. The method has the advantage of using natural detergents, lysophospholipids, which are minor but natural constituents of biological membranes. The method could be of value as a tool in studies of mechanisms of insertion of newly synthesized proteins into biological membranes.
描述了一种使用天然去污剂溶血磷脂酰胆碱或溶血磷脂酸之一将蛋白质重组到脂质小泡中的方法。肠道微绒毛酶氨肽酶N（EC 3.4.11.2）被整合到由微绒毛膜的总脂质提取物制备的脂质小泡中。该方法基于氨肽酶-溶血磷脂胶束与通过超声处理制备的脂质体的融合。通过凝胶渗透色谱法和蔗糖密度梯度离心法分析蛋白质在脂质双层中的整合情况。蛋白质和脂质分布的一致性用于评估蛋白质的整合。通过负染色电子显微镜观察整合情况。该方法的优点是使用天然去污剂溶血磷脂，它们是生物膜中的微量但天然的成分。该方法作为研究新合成蛋白质插入生物膜机制的工具可能具有价值。
