Darmon A, Bar-Noy S, Ginsburg H, Cabantchik Z I
Biochim Biophys Acta. 1985 Jul 25;817(2):238-48. doi: 10.1016/0005-2736(85)90025-2.
The two major membrane glycoproteins of human red cells, glycophorin and band 3, the anion exchange protein, were isolated from cells exofacially labeled with fluorescein and reconstituted into vesicles with defined transmembrane disposition. Uniform orientation of polypeptides was accomplished by two procedures: Vesicles with single protein units were obtained by a one-step dilution of a protein/detergent suspension with a vast excess of phospholipid. Vesicles with uniform orientation of protein were selected by affinity chromatography on derivatized Sepharoses (organomercurial, wheat germ agglutinin, aminoethyl or diethylaminoethyl). Vesicles with multiple protein units with uniform orientation were generated by vectorial immobilization of detergent solubilized proteins on the above affinity matrices and in situ formation of proteoliposomes by detergent substitution for phospholipid. The proteoliposomes were released from the column by addition of excess free ligand. The orientation of band 3 and glycophorin in the reconstituted vesicles was first assessed by immunofluorescence quenching, using anti-fluorescein antibodies, to quantitatively quench fluorescein residues exposed on the outer surface of vesicles. Further assessment was achieved by chromatographing the vesicles through various affinity and ionic matrices. Vesicle populations of higher than 90% homogeneity in protein orientation (right-side-out or inside-out) were obtained with both procedures. The above methods provide a convenient experimental tool for the oriented reconstitution of proteins and the evaluation of their transmembrane disposition.
人类红细胞的两种主要膜糖蛋白，血型糖蛋白和带3蛋白（阴离子交换蛋白），从用荧光素进行细胞外标记的细胞中分离出来，并重组到具有特定跨膜排列的囊泡中。通过两种方法实现多肽的均匀取向：通过用大量过量的磷脂一步稀释蛋白质/去污剂悬浮液获得具有单个蛋白质单元的囊泡。通过在衍生化的琼脂糖（有机汞、麦胚凝集素、氨乙基或二乙氨基乙基）上进行亲和色谱选择具有均匀蛋白质取向的囊泡。通过将去污剂溶解的蛋白质矢量固定在上述亲和基质上，并通过用磷脂替代去污剂原位形成蛋白脂质体，产生具有多个均匀取向的蛋白质单元的囊泡。通过加入过量的游离配体将蛋白脂质体从柱上洗脱下来。首先通过使用抗荧光素抗体进行免疫荧光淬灭来评估重组囊泡中带3蛋白和血型糖蛋白的取向，以定量淬灭暴露在囊泡外表面的荧光素残基。通过使囊泡通过各种亲和和离子基质进行色谱分析来进一步评估。两种方法均获得了蛋白质取向（外翻或内翻）均一性高于90%的囊泡群体。上述方法为蛋白质的定向重组及其跨膜排列的评估提供了一种方便的实验工具。
