Hackmann Yvonne, Joedicke Lisa, Panneels Valérie, Sinning Irmgard
Biochemistry Center, Heidelberg University, Heidelberg, Germany.
Biochemistry Center, Heidelberg University, Heidelberg, Germany.
Methods Enzymol. 2015;556:219-39. doi: 10.1016/bs.mie.2014.12.012. Epub 2015 Mar 6.
In recent years, improved protein expression and crystallization strategies, as well as advanced synchrotron radiation sources and crystallographic tools considerably increased the number of crystal structures of integral membrane proteins from higher eukaryotes. However, seen as a proportion of the total number of candidate proteins, these achievements still appear meager, reflecting the huge effort that is often required to obtain high-level and functional expression of eukaryotic membrane proteins. Besides bacteria, yeast, insect, or mammalian cells are frequently used for heterologous expression, but despite considerable investments in time, labor, and money, there are numerous drawbacks to these systems. Are there other strategies that allow for an effective, large-scale production of functional membrane proteins? This chapter describes the expression of proteins in photoreceptor cells of transgenic Drosophila as an easily accessible, versatile alternative. We present step-by-step protocols starting from the cloning of the target gene into a suitable vector for fly eye expression and ending with the harvest of transgenic Drosophila and subsequent protein purification from the eye. Our examples span a number of eukaryotic membrane proteins from different classes-including receptors, transporters, channels, and enzymes-that were successfully expressed without further optimization. The protocols provided here are robust and straightforward to follow even without prior experience in Drosophila work.
近年来，改进的蛋白质表达和结晶策略，以及先进的同步辐射源和晶体学工具，极大地增加了来自高等真核生物的整合膜蛋白晶体结构的数量。然而，相对于候选蛋白的总数而言，这些成果仍然显得微不足道，这反映出获得真核膜蛋白的高水平功能性表达往往需要付出巨大努力。除了细菌外，酵母、昆虫或哺乳动物细胞也经常用于异源表达，但尽管在时间、人力和资金上投入巨大，这些系统仍存在诸多缺点。是否有其他策略能够实现功能性膜蛋白的有效大规模生产呢？本章介绍了在转基因果蝇的光感受器细胞中表达蛋白质，作为一种易于实现且用途广泛的替代方法。我们提供了详细的步骤方案，从将目标基因克隆到适合果蝇眼睛表达的载体开始，到收获转基因果蝇并随后从眼睛中纯化蛋白质结束。我们的示例涵盖了来自不同类别的多种真核膜蛋白，包括受体、转运蛋白、通道和酶，这些蛋白无需进一步优化即可成功表达。即使没有果蝇研究的先验经验，这里提供的方案也稳健且易于遵循。
