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Cell-free expression and assembly of ATP synthase.

机译：ATP合酶的无细胞表达和装配。

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Cell-free (CF) expression technologies have emerged as promising methods for the production of individual membrane proteins of different types and origin. However, many membrane proteins need to be integrated in complex assemblies by interaction with soluble and membrane-integrated subunits in order to adopt stable and functionally folded structures. The production of complete molecular machines by CF expression as advancement of the production of only individual subunits would open a variety of new possibilities to study their assembly mechanisms, function, or composition. We demonstrate the successful CF formation of large molecular complexes consisting of both membrane-integrated and soluble subunits by expression of the atp operon from Caldalkalibacillus thermarum strain TA2.A1 using Escherichia coli extracts. The operon comprises nine open reading frames, and the 542-kDa F(1)F(o)-ATP synthase complex is composed of 9 soluble and 16 membrane-embedded proteins in the stoichiometry alpha(3)beta(3)gammadeltavarepsilonab(2)c(13). Complete assembly into the functional complex was accomplished in all three typically used CF expression modes by (i) solubilizing initial precipitates, (ii) cotranslational insertion into detergent micelles or (iii) cotranslational insertion into preformed liposomes. The presence of all eight subunits, as well as specific enzyme activity and inhibition of the complex, was confirmed by biochemical analyses, freeze-fracture electron microscopy, and immunogold labeling. Further, single-particle analysis demonstrates that the structure and subunit organization of the CF and the reference in vivo expressed ATP synthase complexes are identical. This work establishes the production of highly complex molecular machines in defined environments either as proteomicelles or as proteoliposomes as a new application of CF expression systems.
机译：无细胞（CF）表达技术已经成为有前途的生产不同类型和来源的单个膜蛋白的方法。然而，许多膜蛋白需要通过与可溶性和膜整合的亚基相互作用而整合成复杂的组装体，以便采用稳定且功能折叠的结构。通过CF表达来生产完整的分子机器，作为仅单个亚基的生产的进步，将为研究其组装机理，功能或组成开辟各种新的可能性。我们展示了大分子复合物的成功CF形成，该复合物由膜整合和可溶亚基组成，通过使用大肠杆菌提取物表达来自Caldalkalibacillus thermarum菌株TA2.A1的atp操纵子来表达。该操纵子包括九个开放阅读框，而542-kDa F（1）F（o）-ATP合酶复合物由化学计量比为alpha（3）beta（3）gammadeltavarepsilonab（2）的9种可溶性和16种膜包埋蛋白组成。 c（13）。通过（i）溶解最初的沉淀物，（ii）共翻译插入去污剂胶束或（iii）共翻译插入预先形成的脂质体，可以在所有三种通常使用的CF表达模式下完成向功能复合物中的完全组装。通过生化分析，冷冻断裂电子显微镜和免疫金标记证实了所有八个亚基的存在，以及特定的酶活性和对复合物的抑制作用。此外，单颗粒分析证明了CF和参考体内表达的ATP合酶复合物的结构和亚基组织是相同的。这项工作建立了在既定环境中作为CF表达系统的新应用的作为蛋白质胶束或作为脂质体的高度复杂分子机器的生产。

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