Cell fractionation

Melissa Petiti (1) , Laetitia Houot (1) , Denis Duché (1)

1 LISM - Laboratoire d'ingénierie des systèmes macromoléculaires

Protein function is generally dependent on its subcellular localisation. 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 localisation within cell such as in silico identification of protein signal sequences and motifs, electron microscopy and immunogold labelling, 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.

Mots clés: Spheroplast; protein solubilisation; subcellular localization.

Domaines: Biochimie, Biologie Moléculaire

Melissa Petiti, Laetitia Houot, Denis Duché. Cell fractionation. Methods in Molecular Biology: Bacterial protein secretion systems, 2017. ⟨hal-01789354⟩