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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., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Protein-protein interaction studies are essential to understand how proteins organize themselves into interaction networks and thus influence cellular processes. Protein binding specificity depends on the correct three-dimensional folding of the polypeptide sequences. One of the forces involved in the structuring and stability of proteins is the formation of disulfide bonds. These covalent bonds are formed posttranscriptionally by the oxidation of a pair of cysteine residues and can serve structural, catalytic, or signaling roles. Here, we describe an engineered E. coli adenylate cyclase mutant strain with an oxidative cytoplasm that promotes correct folding of proteins with disulfide bonds. This genetic background expands the set of host strains suitable for studying protein-protein interactions in vivo by the adenylate cyclase two-hybrid approach., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 a 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.