Proteoliposome Engineering with Cell-Free Membrane Protein Synthesis: Control of Membrane Protein Sorting into Liposomes by Chaperoning Systems.
Proteoliposome Engineering with Cell-Free Membrane Protein Synthesis: Control of Membrane Protein Sorting into Liposomes by Chaperoning Systems.
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使用无细胞膜蛋白合成的蛋白质体工程：通过伴侣系统将膜蛋白分类为脂质体的膜蛋白的控制。
DOI：
10.1002/advs.201800524
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发表时间：
2018-10
期刊：
影响因子：
--
通讯作者：
Akiyoshi K
中科院分区：
文献类型：
--
作者：
Ando M;Schikula S;Sasaki Y;Akiyoshi K
整合膜蛋白（IMP）调节关键的细胞过程;它们的功能障碍与疾病密切相关。然而，活性构象的IMP的生产用于进一步研究受到活表达系统中的聚集和毒性的阻碍。因此，IMP在采用脂质体伴侣的无细胞系统中产生，但新生IMP的膜整合是次优的，并且整合蛋白的方向仍然不可控。因此，基于多组氨酸/镍螯合物亲和力，结合无细胞膜蛋白合成，开发了人工膜蛋白分选系统。其概念证明是用N-末端六组氨酸融合的连接蛋白-43（NHis-Cx43）模型IMP证明的。与Cx43相比，镍螯合脂质体有效地掺入两倍新合成的NHis-Cx43。当在该系统中合成时，NHis-Cx43形成染料可渗透的半通道，类似于细胞中Cx43形成的质膜孔。掺入的NHis-Cx43的拓扑结构表明脂质体膜中的两个取向。然而，NHis-Cx43的取向是受控制的，导致单一的拓扑结构，由天然分子伴侣DnaKJE的组合。使用其他三种NHis融合IMP（包括α螺旋和β桶IMP）也实现了成功合成和至少4.5倍的脂质掺入增加。总的来说，这种简单的膜蛋白分选系统可与分子伴侣组合用于制备用于许多应用的蛋白脂质体。
Integral membrane proteins (IMPs) modulate key cellular processes; their dysfunctions are closely related to disease. However, production of IMPs in active conformations for further study is hindered by aggregation and toxicity in living expression systems. IMPs are therefore produced in cell‐free systems employing liposome chaperoning, but membrane integration of the nascent IMPs is suboptimal and orientation of the integrated proteins remains uncontrollable. Thus, an artificial membrane protein sorting system is developed, based on polyhistidine/nickel‐chelate affinity, combined with cell‐free membrane protein synthesis. Its proof of concept is demonstrated with a N‐terminal hexahistadine‐fused conexin‐43 (NHis–Cx43) model IMP. Nickel‐chelating liposomes efficiently incorporate twofold newly synthesized NHis–Cx43 compared with Cx43. NHis–Cx43, when synthesized in this system, forms dye‐permeable hemichannels, similar to plasma membrane pores formed by Cx43 in cells. The topology of incorporated NHis–Cx43 indicates two orientations in the liposomal membranes. However, NHis–Cx43 orientation is controlled, resulting in single topology, by combination of the natural molecular chaperone DnaKJE. Successful synthesis and at least 4.5‐fold increase lipid incorporation are also achieved with three other NHis‐fused IMPs, including α‐helix and β‐barrel IMPs. Overall, this simple membrane protein sorting system is usable combined with molecular chaperones to prepare proteoliposomes for many applications.
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影响因子：
4.6
作者：
Ohta N;Kato Y;Watanabe H;Mori H;Matsuura T
通讯作者：
Matsuura T
DOI：
10.1038/nrm3226
发表时间：
2011-11-16
期刊：
Nature reviews. Molecular cell biology
影响因子：
--
作者：
通讯作者：
--
影响因子：
5.4
作者：
Almen, Markus Sallman;Nordstrom, Karl J. V.;Schioth, Helgi B.
通讯作者：
Schioth, Helgi B.
影响因子：
16.6
作者：
Matsubayashi, Hideaki;Kuruma, Yutetsu;Ueda, Takuya
通讯作者：
Ueda, Takuya
影响因子：
3.4
作者：
Aschtgen, Marie-Stephanie;Zoued, Abdelrahim;Lloubes, Roland;Journet, Laure;Cascales, Eric
通讯作者：
Cascales, Eric