Different protein localizations on the inner and outer leaflet of cell-sized liposomes using cell-free protein synthesis
Different protein localizations on the inner and outer leaflet of cell-sized liposomes using cell-free protein synthesis
复制标题
使用无细胞蛋白质合成在细胞大小的脂质体的内部和外部小叶上进行不同的蛋白质定位
DOI：
10.1093/synbio/ysy007
复制
发表时间：
2018
影响因子：
3.2
通讯作者：
Matsuura Tomoaki
中科院分区：
文献类型：
--
作者：
Uyeda Atsuko;Watanabe Takayoshi;Hohsaka Takahiro;Matsuura Tomoaki
活细胞的膜具有不对称性。膜的内叶和外叶由不同的磷脂组成，它们影响膜蛋白的功能，而这种不对称性的丧失被认为会导致细胞凋亡。此外，在膜的内外小叶上发现了不同的蛋白质，它们是各种生化反应所必需的，包括与信号转导和细胞形态有关的反应。尽管磷脂组成不对称的玻璃体脂双层重构已有报道，但不同蛋白质定位于内、外叶的脂双层的重构仍然很难实现蛋白质的不对称定位。在这里，我们开发了一种简单的方法来实现这种不对称使用在试管转录-翻译系统(IVTT)。该方法使用了苯基鸟嘌呤(BG)衍生物修饰的磷脂，该磷脂与Snap-Tag序列形成共价键。我们发现，通过BG和Snap-Tag之间的相互作用，纯化的Snap-tag蛋白可以定位到细胞大小的脂质体表面。然后我们发现IVTT合成的蛋白质可以定位在脂膜上，不同的蛋白质可以不对称地定位在脂质体的外叶和内叶上。
Membranes of living cells possess asymmetry. The inner and outer leaflets of the membrane consist of different phospholipid compositions, which are known to affect the function of membrane proteins, and the loss of the asymmetry has been reported to lead to cell apoptosis. In addition, different proteins are found on the inner and outer leaflets of the membrane, and they are essential for various biochemical reactions, including those related to signal transduction and cell morphology. Whilein vitrolipid bilayer reconstitution with asymmetric phospholipid compositions has been reported, the reconstitution of lipid bilayer where different proteins are localized in the inner and outer leaflet, thereby enables asymmetric protein localizations, has remained difficult. Herein, we developed a simple method to achieve this asymmetry using anin vitrotranscription–translation system (IVTT). The method used a benzylguanine (BG) derivative-modified phospholipid, which forms a covalent bond with a snap-tag sequence. We show that purified snap-tagged protein can be localized to the cell-sized liposome surface via an interaction between BG and the snap-tag. We then show that IVTT-synthesized proteins can be located at the lipid membrane and that different proteins can be asymmetrically localized on the outer and inner leaflets of liposomes.
登录
查看更多内容
通过脂质体展示进行体外膜蛋白工程
DOI：
--
发表时间：
2017
期刊：
影响因子：
--
作者：
Kosuke Noguchi;Ayako Aoki;Tomoka Takatani-Nakase;Ikuo Fujii;Shiroh Futaki;Ikuhiko Nakase;Tomoaki Matsuura
通讯作者：
Tomoaki Matsuura
影响因子：
3.9
作者：
Nishimura, Kazuya;Hosoi, Tomohiro;Yomo, Tetsuya
通讯作者：
Yomo, Tetsuya
DOI：
10.1016/j.bbamem.2008.01.023
发表时间：
2008-05
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Jessica J Wuu;J. Swartz
通讯作者：
Jessica J Wuu;J. Swartz
影响因子：
4.8
作者：
Shimizu, Y;Kanamori, T;Ueda, T
通讯作者：
Ueda, T
影响因子：
16
作者：
Carlson, Erik D.;Gan, Rui;Hodgman, C. Eric;Jewett, Michael C.
通讯作者：
Jewett, Michael C.