Henrich Erik, Dötsch Volker, Bernhard Frank
Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J.W. Goethe-University, Frankfurt-am-Main, Germany.
Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J.W. Goethe-University, Frankfurt-am-Main, Germany.
Methods Enzymol. 2015;556:351-69. doi: 10.1016/bs.mie.2014.12.016. Epub 2015 Mar 20.
Cell-free (CF) protein expression has emerged as one of the most efficient production platforms for membrane proteins. Central bottlenecks prevalent in conventional cell-based expression systems such as mistargeting, inclusion body formation, degradation as well as product toxicity can be addressed by taking advantage of the reduced complexity of CF expression systems. However, the open accessibility of CF reactions offers the possibility to design customized artificial expression environments by supplying synthetic hydrophobic compounds such as micelles or membranes of defined composition. The open nature of CF systems therefore generally allows systematic screening approaches for the identification of efficient cotranslational solubilization environments of membrane proteins. Synergies exist in particular with the recently developed nanodisc (ND) technology enabling the synthesis of stable and highly soluble particles containing membrane discs of defined composition. Specific types of lipids frequently modulate folding, stability, and activity of integrated membrane proteins. One recently reported example are phospho-MurNAc-pentapeptide (MraY) translocases that catalyze a crucial step in bacterial peptidoglycan biosynthesis making them interesting as future drug targets. Production of functionally active MraY homologues from most human pathogens in conventional cellular production systems was so far not successful due to their obviously strict lipid dependency for functionally folding. We demonstrate that the combination of CF expression with ND technologies is an efficient strategy for the production of folded MraY translocases, and we present a general protocol for the rapid screening of lipid specificities of membrane proteins.
无细胞（CF）蛋白质表达已成为膜蛋白最有效的生产平台之一。传统基于细胞的表达系统中普遍存在的核心瓶颈，如错误靶向、包涵体形成、降解以及产物毒性等问题，可以通过利用CF表达系统降低的复杂性来解决。然而，CF反应的开放性使得通过提供合成疏水化合物（如具有特定组成的胶束或膜）来设计定制的人工表达环境成为可能。因此，CF系统的开放性通常允许采用系统筛选方法来鉴定膜蛋白有效的共翻译溶解环境。特别是与最近开发的纳米盘（ND）技术存在协同作用，该技术能够合成含有特定组成膜盘的稳定且高度可溶的颗粒。特定类型的脂质经常调节整合膜蛋白的折叠、稳定性和活性。最近报道的一个例子是磷酸化MurNAc - 五肽（MraY）转位酶，它催化细菌肽聚糖生物合成中的关键步骤，使其作为未来的药物靶点很有吸引力。由于大多数人类病原体的功能性MraY同源物在功能折叠上明显严格依赖脂质，因此到目前为止在传统细胞生产系统中生产具有功能活性的MraY同源物并不成功。我们证明CF表达与ND技术的结合是生产折叠的MraY转位酶的有效策略，并且我们提出了一种用于快速筛选膜蛋白脂质特异性的通用方案。
