Berger Imre, Garzoni Frederic, Chaillet Maxime, Haffke Matthias, Gupta Kapil, Aubert Alice
J Vis Exp. 2013 Jul 11(77):e50159. doi: 10.3791/50159.
Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.(1,2) Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.(3) BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.(4) A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.(5-8) The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.
蛋白质组学研究以前所未有的细节揭示了真核生物蛋白质组令人惊叹的复杂性。现在人们普遍认为，细胞中的蛋白质大多并非以孤立的实体形式存在，而是与许多其他蛋白质相互作用发挥其生物学活性，在人类细胞中这种相互作用的蛋白质有十种或更多，它们在细胞中形成装配线以执行大部分（如果不是全部）重要功能。（1,2）要了解这些多蛋白装配体的功能和结构，需要以高质量和足够的数量提供它们，以便进行详细分析。细胞中许多蛋白质复合物数量稀少，尤其是在真核生物中，这使得无法从天然来源提取它们，因此需要进行重组生产。杆状病毒表达载体系统（BEVS）已被证明在生产真核蛋白质方面特别有用，因为真核蛋白质的活性通常依赖于翻译后加工，而其他常用的表达系统往往无法支持这种加工。（3）BEVS使用插入了感兴趣基因的重组杆状病毒来感染昆虫细胞培养物，昆虫细胞培养物进而产生所需的蛋白质。MultiBac是一种专门为生产包含多个亚基的真核蛋白质复合物而设计的BEVS。（4）高效生产蛋白质及其复合物的一个至关重要的前提是，表达实验中涉及的所有步骤都要有稳健的方案，理想情况下这些方案可以作为标准操作规程（SOPs）实施，并且非专业用户也能相对轻松地遵循。欧洲分子生物学实验室（EMBL）的MultiBac平台在多蛋白复合物表达实验的所有步骤中都使用SOPs，从将基因插入针对异源蛋白质生产特性进行优化的工程杆状病毒基因组，到对所产生的蛋白质样本进行小规模分析。（5 - 8）该平台以开放获取的模式安装在EMBL格勒诺布尔，已经支持了许多来自学术界和工业界的科学家加速蛋白质复合物研究项目。
