Sedzik Jan, Uyemura Keiichi, Tsukihara Tomitake
Institute for Protein Research, Osaka University, Osaka 565-0871, Japan.
Protein Expr Purif. 2002 Dec;26(3):368-77. doi: 10.1016/s1046-5928(02)00564-8.
The preparation of a pure and homogeneous protein sample at proper concentration is a prerequisite for success when attempting their crystallization for structural determination. The detergents suitable for solubilization particularly of membrane proteins are not always the best for crystallization. Myelin of the peripheral nervous system of vertebrates is the example of a membrane for which neutral or "gentle" detergents are not even strong enough to solubilize its proteins. In contrast, sodium- or lithium-dodecyl sulfate is very effective. We solubilized myelin membrane in 2%(w/v) sodium dodecyl sulfate, followed by chromatographic purification of the hydrophobic myelin glycoproteins P0 and PASII/PMP22, and finally, we have exchanged the sodium dodecyl sulfate bound to protein for other neutral detergents using ceramic hydroxyapatite column. Theoretically, we should easily exchange sodium dodecyl sulfate for any neutral detergent, but for some of them, the solubility of myelin glycoproteins is low. To monitor the potential variability in the secondary structure of glycoproteins, we have used circular dichroism. Sodium dodecyl sulfate seems to be the appropriate detergent for the purpose of purification of very hydrophobic glycoproteins, since it can be easily exchanged for another neutral detergent.
为了进行结构测定而尝试对蛋白质进行结晶时，制备浓度合适的纯且均匀的蛋白质样品是成功的前提条件。适合溶解尤其是膜蛋白的去污剂并不总是最适合结晶的。脊椎动物外周神经系统的髓磷脂就是一个例子，对于这种膜，中性或“温和”的去污剂甚至不足以溶解其蛋白质。相比之下，十二烷基硫酸钠或十二烷基硫酸锂非常有效。我们用2%（w/v）的十二烷基硫酸钠溶解髓磷脂膜，随后对疏水性髓磷脂糖蛋白P0和PASII/PMP22进行色谱纯化，最后，我们使用陶瓷羟基磷灰石柱将与蛋白质结合的十二烷基硫酸钠换成其他中性去污剂。理论上，我们应该很容易将十二烷基硫酸钠换成任何中性去污剂，但对于其中一些去污剂，髓磷脂糖蛋白的溶解度较低。为了监测糖蛋白二级结构的潜在变化，我们使用了圆二色性。十二烷基硫酸钠似乎是用于纯化非常疏水的糖蛋白的合适去污剂，因为它可以很容易地换成另一种中性去污剂。
