Isolation of an integral membrane glycoprotein by chloroform-methanol extraction and C3-reversed-phase high-performance liquid chromatography.
Isolation of an integral membrane glycoprotein by chloroform-methanol extraction and C3-reversed-phase high-performance liquid chromatography.
复制标题
通过氯仿-甲醇萃取和 C3 反相高效液相色谱分离完整膜糖蛋白。
DOI：
10.1016/0003-2697(87)90521-5
复制
发表时间：
1987
影响因子：
2.9
通讯作者：
Poduslo,JF
中科院分区：
文献类型：
--
作者：
Brunden,KR;Berg,CT;Poduslo,JF
介绍了一种分离完整膜蛋白的方法，并将其应用于主要髓鞘糖蛋白P0的纯化。这一分离方案依赖于从坐骨神经内神经细胞中等渗提取的膜部分的洗涤剂增溶，然后通过氯仿/甲醇提取去除脂质和洗涤剂。所得膜蛋白易溶于醋酸/水(1 1)，可直接用反相高效液相色谱分析。固有的膜蛋白混合物的疏水性导致与C8固定相的强烈结合，导致较差的拆分和产率。这些问题可以通过使用C3烷基硅烷柱来消除，从而允许分离蛋白质组分和分离P0。纯化的P0有一个氨基端序列，与核苷酸测序预测的序列相匹配，糖蛋白含有预期的唾液酸量。这一发现表明，分离过程不会损害P0的复合型低聚糖结构，并应使该方法易于应用于其他完整膜蛋白和糖蛋白的纯化。
Methodology is presented for the isolation of integral membrane proteins and applied to the purification of the major myelin glycoprotein, P0. This isolation scheme depends on the detergent solubilization of an isoosmotically extracted membrane fraction from sciatic nerve endoneurium, followed by the removal of lipids and detergent by chloroform/methanol extraction. The resulting membrane proteins are readily dissolved in acetic acid/water (1 1 ) and directly analyzed by reversed-phase high-performance liquid chromatography. The hydrophobic nature of the intrinsic membrane protein mixture results in strong binding to a C8stationary phase, leading to poor resolution and yields. These problems can be eliminated by employing a C3alkylsilane column, thereby allowing separation of the protein components and the isolation of P0. The purified P0has an amino-terminal sequence that matches that predicted from nucleotide sequencing, and the glycoprotein contains the expected amount of sialic acid. This latter finding indicates that the isolation procedure is not detrimental to the complex-type oligosaccharide structure of P0and should make the methodology readily applicable to the purification of other integral membrane proteins and glycoproteins.