Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system.
Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system.
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DOI：
10.1186/1472-6750-11-35
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发表时间：
2011-04-11
影响因子：
3.5
通讯作者：
Endo Y
中科院分区：
文献类型：
--
作者：
Nozawa A;Ogasawara T;Matsunaga S;Iwasaki T;Sawasaki T;Endo Y
最近，一些研究小组报道了在外源性脂质体（脂质体）存在下无细胞合成功能性膜蛋白（MP）。在此之前，我们报道了合成的功能AtPPT 1植物磷酸盐转运蛋白，与脂质体在翻译过程中。然而，目前还不清楚脂质/MP复合物的形成是否是常见的所有类型的MP在小麦无细胞系统。AtPPT 1的合成使用小麦无细胞系统与或没有脂质体。用脂质体合成的AtPPT 1具有较高的转运活性，但不加脂质体合成的AtPPT 1的转运活性不到加脂质体合成的AtPPT 1的10%。为了测试在其他MP的合成中是否观察到与脂质体的共翻译关联，我们使用了具有1至14个跨膜结构域（TMD）的40种哺乳动物MP和5种可溶性蛋白作为对照。40种MP与脂质体的结合率均大于40%（平均值为59%），而5种可溶性蛋白的结合率均小于20%（平均值为12%）。无论TMD的数量和合成产率如何，MP之间的结合率都没有显着差异。这些结果表明，小麦无细胞系统是一种高产率的脂质/MP复合物形成方法，适合于大规模制备。还测试了绿色荧光蛋白（GFP）融合MP的脂质体缔合，并在通过Accudenz密度梯度超离心（DGU）浮选后作为脂质/MP复合物回收。GFP-MP的使用揭示了Accudenz浮选的最佳条件。使用优化的Accudenz DGU条件，部分纯化P2 RX 4/脂质复合物，并在SDS-PAGE后通过考马斯亮蓝（CBB）染色检测为主带。在无细胞合成反应期间脂质/AtPPT 1复合物的形成对于功能性MP的合成至关重要。在所有40个测试的MP中观察到翻译期间的脂质/MP复合物。至少29个MP，根据其与GFP相比更高的生产率判断，可能适合于大规模制备。通过该方法合成的MP形成脂质/MP复合物，其可以容易地通过Accudenz DGU部分纯化。利用脂质体合成小麦无细胞蛋白质将为制备多种类型的MP提供一种有效的方法。
Recently, some groups have reported on cell-free synthesis of functional membrane proteins (MPs) in the presence of exogenous liposomes (liposomes). Previously, we reported synthesis of a functional AtPPT1 plant phosphate transporter that was associated with liposomes during translation. However, it is unclear whether or not lipid/MP complex formation is common to all types of MPs in the wheat cell-free system. AtPPT1 was synthesized using a wheat cell-free system with or without liposomes. AtPPT1 synthesized with liposomes showed high transport activity, but the activity of AtPPT1 synthesized without liposomes was less than 10% activity of that with liposomes. To test whether co-translational association with liposomes is observed in the synthesis of other MPs, we used 40 mammalian MPs having one to 14 transmembrane domains (TMDs) and five soluble proteins as a control. The association rate of all 40 MPs into liposomes was more than 40% (mean value: 59%), while that of the five soluble proteins was less than 20% (mean value: 12%). There were no significant differences in association rate among MPs regardless of the number of TMDs and synthesis yield. These results indicate that the wheat cell-free system is a highly productive method for lipid/MP complex formation and is suitable for large-scale preparation. The liposome association of green fluorescent protein (GFP)-fusion MPs were also tested and recovered as lipid/MP complex after floatation by Accudenz density gradient ultracentrifugation (DGU). Employment of GFP-MPs revealed optimal condition for Accudenz floatation. Using the optimized Accudenz DGU condition, P2RX4/lipid complexes were partially purified and detected as a major band by Coomassie Brilliant Blue (CBB)-staining after SDS-PAGE. Formation of lipid/AtPPT1 complex during the cell-free synthesis reaction is critical for synthesis of a functional MP. The lipid/MP complex during the translation was observed in all 40 MPs tested. At least 29 MPs, as judged by their higher productivity compared to GFP, might be suitable for a large-scale preparation. MPs synthesized by this method form lipid/MP complexes, which could be readily partially purified by Accudenz DGU. Wheat cell-free protein synthesis in the presence of liposomes will be a useful method for preparation of variety type of MPs.
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DOI：
10.1073/pnas.0804766105
发表时间：
2008-10-14
影响因子：
11.1
作者：
Kaiser, Liselotte;Graveland-Bikker, Johanna;Zhang, Shuguang
通讯作者：
Zhang, Shuguang
影响因子：
4.4
作者：
Katzen, Federico;Fletcher, Julia E.;Kudlicki, Wieslaw
通讯作者：
Kudlicki, Wieslaw
影响因子：
2.9
作者：
Berrier, C;Park, KH;Ghazi, A
通讯作者：
Ghazi, A
影响因子：
46.9
作者：
Shimizu, Y;Inoue, A;Ueda, T
通讯作者：
Ueda, T
影响因子：
2.9
作者：
GRAHAM, JM;FORD, T;RICKWOOD, D
通讯作者：
RICKWOOD, D