Abstract：
Protein function is generally dependent on its subcellular localization. In gram-negative bacteria such as Escherichia coli, a protein can be targeted to five different compartments: the cytoplasm, the inner membrane, the periplasm, the outer membrane, and the extracellular medium. Different approaches can be used to determine the protein localization within cell such as in silico identification of protein signal sequences and motifs, electron microscopy and immunogold labeling, optical fluorescence microscopy, and biochemical technics. In this chapter, we describe a simple and efficient method to isolate the different compartments of Escherichia coli by a fractionation method and to determine the presence of the protein of interest. For inner membrane proteins, we propose a method to discriminate between integral and peripheral membrane proteins.
摘要：
蛋白质功能通常取决于其亚细胞定位。在革兰氏阴性菌如大肠杆菌中，蛋白质可以靶向五个不同的区室：细胞质，内膜，周质，外膜，和细胞外培养基。可以使用不同的方法来确定细胞内的蛋白质定位，例如蛋白质信号序列和基序的计算机识别，电子显微镜和免疫金标记，光学荧光显微镜，和生化技术。在这一章中,我们描述了一种简单有效的方法，通过分级分离方法分离大肠杆菌的不同区室，并确定感兴趣的蛋白质的存在。对于内膜蛋白，我们提出了一种区分完整膜蛋白和外周膜蛋白的方法。
