Seppen J, Jansen P L, Oude Elferink R P
Department of Gastrointestinal and Liver Diseases, Academic Medical Centre, Amsterdam, The Netherlands.
Protein Expr Purif. 1995 Apr;6(2):149-54. doi: 10.1006/prep.1995.1018.
When membrane proteins are solubilized and subjected to purification procedures, the loss of lipids surrounding the protein often results in irreversible inactivation. We describe a procedure for the immunoaffinity purification of the membrane protein UDP-glucuronosyltransferase from human liver. This procedure reduces exposure of the protein to detergent, thereby reducing lipid loss. Triton X-100 was used to solubilize human liver microsomes. The solubilized proteins were applied to a sephacryl 300 (S-300) gel filtration column equilibrated with detergent-free buffer. UDP-glucuronosyltransferase activity eluted in turbid fractions in the void volume. During passage through the column Triton X-100 can partition in the buffer, while lipids are reconstituted into vesicular structures. Proteins with a high affinity for phospholipids remain associated with the lipid and elute in the void volume. The active fractions from the S-300 column were resolubilized with Triton X-100 and applied to a column with immobilized antibody. Washing this column with buffer containing phosphatidylcholine liposomes and no detergent removed unbound proteins and minimized loss of lipid from bound UDP-glucuronosyltransferase. Raising the pH of the washing buffer to 11.5 in the presence of liposomes resulted in elution and simultaneous reconstitution of active UDP-glucuronosyltransferase. Antibodies against membrane proteins are often available but immunoaffinitypurification of active enzyme is difficult. The approach described here could be useful for the isolation of other membrane proteins.
当膜蛋白被溶解并进行纯化时，蛋白质周围脂质的丧失常常导致不可逆的失活。我们描述了一种从人肝脏免疫亲和纯化膜蛋白UDP-葡糖醛酸基转移酶的方法。该方法减少了蛋白质与去污剂的接触，从而减少了脂质的损失。使用Triton X-100溶解人肝脏微粒体。将溶解的蛋白质应用于用无去污剂缓冲液平衡的Sephacryl 300（S-300）凝胶过滤柱。UDP-葡糖醛酸基转移酶活性在空体积的浑浊级分中洗脱。在通过柱子的过程中，Triton X-100可在缓冲液中分配，而脂质则重新组装成囊泡结构。对磷脂具有高亲和力的蛋白质与脂质保持结合并在空体积中洗脱。将来自S-300柱的活性级分用Triton X-100重新溶解，并应用于固定化抗体柱。用含有磷脂酰胆碱脂质体且无去污剂的缓冲液洗涤该柱，可去除未结合的蛋白质，并使结合的UDP-葡糖醛酸基转移酶的脂质损失最小化。在脂质体存在下将洗涤缓冲液的pH提高到11.5，导致活性UDP-葡糖醛酸基转移酶洗脱并同时重新组装。针对膜蛋白的抗体通常是可获得的，但活性酶的免疫亲和纯化很困难。这里描述的方法可能有助于分离其他膜蛋白。
