Saarenpää Tuulia, Jaakola Veli-Pekka, Goldman Adrian
Department of Biochemistry, Helsinki University, Helsinki, Finland.
Novartis Institutes of Biomedical Research, Basel, Switzerland.
Methods Enzymol. 2015;556:185-218. doi: 10.1016/bs.mie.2014.12.033. Epub 2015 Mar 21.
G-protein-coupled receptors (GPCRs) are a large family of seven transmembrane proteins that influence a considerable number of cellular events. For this reason, they are one of the most studied receptor types for their pharmacological and structural properties. Solving the structure of several GPCR receptor types has been possible using almost all expression systems, including Escherichia coli, yeast, mammalian, and insect cells. So far, however, most of the GPCR structures solved have been done using the baculovirus insect cell expression system. The reason for this is mainly due to cost-effectiveness, posttranslational modification efficiency, and overall effortless maintenance. The system has evolved so much that variables starting from vector type, purification tags, cell line, and growth conditions can be varied and optimized countless ways to suit the needs of new constructs. Here, we present the array of techniques that enable the rapid and efficient optimization of expression steps for maximal protein quality and quantity, including our emendations.
G蛋白偶联受体（GPCRs）是一类由七个跨膜蛋白组成的大家族，它们影响着大量的细胞活动。因此，就其药理和结构特性而言，它们是研究最多的受体类型之一。使用几乎所有的表达系统，包括大肠杆菌、酵母、哺乳动物细胞和昆虫细胞，都有可能解析几种GPCR受体类型的结构。然而，到目前为止，大多数已解析的GPCR结构都是使用杆状病毒昆虫细胞表达系统完成的。其主要原因在于成本效益、翻译后修饰效率以及总体上易于维护。该系统已经发展得非常完善，从载体类型、纯化标签、细胞系到生长条件等变量都可以以无数种方式进行改变和优化，以满足新构建体的需求。在这里，我们展示了一系列能够快速有效地优化表达步骤以实现最大蛋白质质量和产量的技术，包括我们的改进方法。
