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.

中文翻译：

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无细胞膜蛋白合成的蛋白脂质体工程：通过陪伴系统控制膜蛋白分选到脂质体中

整合膜蛋白 (IMP) 调节关键的细胞过程；他们的功能障碍与疾病密切相关。然而，用于进一步研究的活性构象 IMP 的生产受到活体表达系统中聚集和毒性的阻碍。因此，IMP 是在采用脂质体陪伴的无细胞系统中产生的，但新生 IMP 的膜整合不是最理想的，并且整合蛋白质的方向仍然无法控制。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，开发了一种人工膜蛋白分选系统。其概念验证通过 N 端六组氨酸融合的 conxin-43 (NHis-Cx43) 模型 IMP 进行了演示。与 Cx43 相比，镍螯合脂质体可以有效地掺入两倍新合成的 NHis-Cx43。NHis-Cx43 在该系统中合成时，形成可渗透染料的半通道，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有两个方向。然而，NHis-Cx43 方向受到控制，通过天然分子伴侣 DnaKJE 的组合产生单一拓扑。其他三种 NHis 融合 IMP（包括 α 螺旋和 β 桶 IMP）也成功合成并增加了至少 4.5 倍的脂质掺入。总体而言，这种简单的膜蛋白分选系统可与分子伴侣结合使用来制备用于多种应用的蛋白脂质体。