Novartis Institutes for BioMedical Research, Basel, Switzerland.
Adv Exp Med Biol. 2012;992:179-96. doi: 10.1007/978-94-007-4954-2_10.
Target proteins in contemporary NMR are becoming increasingly complex. The interest lies on membrane proteins, multi-domain proteins, secreted proteins with secondary modifications etc., which can often only be expressed in eukaryotic expression hosts. Although E. coli is the organism of choice for expression of proteins in isotope labeled form for NMR studies, bacterial cells have limitations concerning their ability of producing soluble and well-folded proteins of human origin. Insect cells are eukaryotic cells and therefore evolutionary closer to human cells than bacterial cells. Therefore a larger share of human proteins can be functionally expressed in them, for example multi-domain proteins, protein complexes, membrane proteins and proteins requiring post-translational modifications. In order to study these proteins by NMR, they are ideally prepared in isotope labeled form. In this chapter different strategies for isotope labeling in insect cells are described - uniform and amino acid specific. A general introduction to expression with baculovirus infected insect cells is given followed by a detailed descriptions of labeling approaches. The chapter is concluded with case studies, describing successful application of isotope labeling in insect cells for NMR studies including solid-state experiments, ligand binding studies and protein dynamics.
当前 NMR 的目标蛋白变得越来越复杂。人们的兴趣主要集中在膜蛋白、多结构域蛋白、具有二级修饰的分泌蛋白等，这些蛋白通常只能在真核表达宿主中表达。尽管大肠杆菌是用于 NMR 研究的同位素标记形式的蛋白质表达的首选生物体，但细菌细胞在产生可溶性和折叠良好的人类来源的蛋白质方面存在局限性。昆虫细胞是真核细胞，因此与细菌细胞相比，它们在进化上更接近人类细胞。因此，更大比例的人类蛋白可以在其中得到功能性表达，例如多结构域蛋白、蛋白复合物、膜蛋白和需要翻译后修饰的蛋白。为了通过 NMR 研究这些蛋白，它们理想地以同位素标记的形式进行制备。本章描述了昆虫细胞中同位素标记的不同策略——均一标记和氨基酸特异性标记。首先概述了杆状病毒感染的昆虫细胞表达，然后详细描述了标记方法。本章以案例研究结束，描述了在 NMR 研究中使用昆虫细胞进行同位素标记的成功应用，包括固态实验、配体结合研究和蛋白动力学。
