Fractionating Cells

Fractionating Escherichia coli cells in a periplasmic fraction and a cytoplasmic fraction by a lysozyme/EDTA – osmotic shock combined method. Fractionating buffer: o o o o o o o o

200mM Tris-HCl pH7.5 0,5mg/ml lysozyme 1mM EDTA 20% sucrose 0,2%(v/v) Triton X-100 50mM benzamidine 0,1mM PMSF 0,5mg/ml iodoacetamide

Wash solution: o

50mM Tris-HCl pH7,5

Protocol -

Centrifuge samples in falcon tubes at 13 000g for 15 minutes at 4°C. Remove the supernatant (which is the extracellular fraction) Resuspend the pellet in wash solution (same volume as original volume of sample) Centrifuge again at 13 000g for 15 minutes at 4°C Discard the supernatant containing remaining growth media Resuspend the pellet in fractionating buffer: 1/5 of the original culture volume Incubate the cell suspension statically at room temperature for 15 minutes Add an equal amount of cold (4°C) dH2O Incubate the cell suspension statically at room temperature for another 15 minutes Centrifuge the cell suspension at 13 000g for 15 minutes at 4°C The supernatant is the periplasmic fraction, transfer this fraction to another labelled falcon tube or Eppendorf tube and store at 4°C or freeze at -20°C Resuspend the pellet in fractionating buffer: 1/5 of the original culture volume Sonicate cells (preferably with a sonicator-probe): 6 x 10s bursts with 10s intervals. This lysed fraction is the cytoplastic fraction. Store this fraction at 4°C upon use or freeze at -20°C. Note: in case of soluble recombinant proteins, this fraction can be centrifuged, since the supernatant will then contain the rec. proteins. In case of inclusion bodies, do not centrifuge because the pellet will contain the proteins of interest. Possibly solubilize the inclusion bodies by mild solubilization agents like 2-propanol (5%v/v)

Sources This protocol is based on following scientific articles: Birdsell, D. C., & Cota-Robles, E. H. (1966). Production and Ultrastructure if Lysozyme and Ethylenediaminetetraacetate-Lysozyme Spheroplasts of Escherichia coli. Riverside, California: Journal of Bacteriology.

French, C., Keshavarz-Moore, E., & Ward, J. M. (1996). Development of a simple method for the recovery of recominant proteins from the Escherichia coli periplasm. New York: Elsevier Science. Jalalirad, R. (2013). Selective and efficient extraction of recombinant proteins from the periplasm of Escherichia coli using low concentrations of chemicals. Birmingham, UK: Springer. McCormack, C. C., Hobson , A. H., Doyle, S., Jackson, J., Kilty, C., & Whitehead, A. S. (1996). Generation of soluble recombinant human acute phase serum amyloid A2 (A-SAA2) protein and its use in development of a A-SAA specific EILSA. Dublin, Ireland: Elsevier Science. Voss, J. G. (1964). Lysozyme Lysis of Gram-Negative Bacteria without Production of Spheroplasts. UK: Journal of General Microbiology.