Cell Fractionation

Part of the Methods in Molecular Biology book series (MIMB, volume 1615)

Abstract

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.

Key words

Spheroplast Peptidoglycan Osmotic shock Freeze and thaw Protein solubilisation Membrane Subcellular localisation 

Notes

Acknowledgments

This work was supported by the Centre National de la Recherche Scientifique and the Agence National de la Recherche (ANR-14-CE09-0023).

References

  1. 1.
    Kaback HR (1972) Transport across isolated bacterial cytoplasmic membranes. Biochim Biophys Acta 265:367–416CrossRefGoogle Scholar
  2. 2.
    Kellenberger E, Ryther A (1958) Cell wall and cytoplasmic membrane of Escherichia coli. J Biophys Biochem Cytol 25:323–326CrossRefGoogle Scholar
  3. 3.
    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
  4. 4.
    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
  5. 5.
    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
  6. 6.
    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
  7. 7.
    Kaback HR (1971) Bacterial membranes. Methods Enzymol 22:99–120Google Scholar
  8. 8.
    Witholt B, Heerikhuizen HV, De Leij L (1976) How does lysozyme penetrate through the bacterial outer membrane. Biochim Biophys Acta 443:534–544CrossRefGoogle Scholar
  9. 9.
    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
  10. 10.
    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
  11. 11.
    Schnaitman CA (1971) Solubilization of the cytoplasmic membrane of Escherichia coli by triton X-100. J Bacteriol 108:545–552PubMedPubMedCentralGoogle Scholar
  12. 12.
    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

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 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

Personalised recommendations