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.
Spheroplast Peptidoglycan Osmotic shock Freeze and thaw Protein solubilisation Membrane Subcellular localisation
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
This work was supported by the Centre National de la Recherche Scientifique and the Agence National de la Recherche (ANR-14-CE09-0023).
Kaback HR (1972) Transport across isolated bacterial cytoplasmic membranes. Biochim Biophys Acta 265:367–416CrossRefGoogle Scholar
Kellenberger E, Ryther A (1958) Cell wall and cytoplasmic membrane of Escherichia coli. J Biophys Biochem Cytol 25:323–326CrossRefGoogle Scholar
Neu HC, Heppel LA (1964) The release of Ribonuclease into the medium when Escherichia coli cells are converted to spheroplasts. J Biol Chem 239:3893–3900PubMedGoogle Scholar
French C, Keshavarz-Moore E, Ward JM (1996) Development of a simple method for the recovery of recombinant proteins from the Escherichia coli periplasm. Enzym Microb Technol 19:332–338CrossRefGoogle Scholar
Skerra A, Plückthun A (1991) Secretion and in vivo folding of the Fab fragment of the antibody McPC603 in Escherichia coli: influence of disulphides and cis-prolines. Protein Eng 4:971–979CrossRefGoogle Scholar
Nossal NG, Heppel LA (1966) The release of enzymes by osmotic shock from Escherichia coli in exponential phase. J Biol Chem 241:3055–3062PubMedGoogle Scholar
Witholt B, Heerikhuizen HV, De Leij L (1976) How does lysozyme penetrate through the bacterial outer membrane. Biochim Biophys Acta 443:534–544CrossRefGoogle Scholar
Mowbray J, Moses V (1976) The tentative identification in Escherichia coli of a multienzyme complex with glycolytic activity. Eur J Biochem 66:25–36CrossRefGoogle Scholar
Schook W, Puszkin P, Bloom W, Ores C, Kochwa S (1979) Mechanochemical properties of brain clathrin: interactions with actin and alpha-actinin and polymerization into basketlike structures or filaments. Proc Natl Acad Sci U S A 76:116–120CrossRefGoogle Scholar
Fujiki Y, Fowler S, Shio H, Hubbard AL, Lazarow PB (1982) Polypeptide and phospholipid composition of the membrane of rat liver peroxisomes: comparison with endoplasmic reticulum and mitochondrial membranes. J Cell Biol 93:103–110CrossRefGoogle Scholar
1.Laboratoire d’Ingénierie des Systèmes Macromoléculaires (LISM, UMR 7255)Institut de Microbiologie de la Méditerranée (IMM), Aix-Marseille Université—Centre National de la Recherche Scientifique (CNRS)Marseille Cedex 20France