Recovering secreted proteins from E. coli periplasm
June 11, 2009
1. Cells are harvested by centrifugation at 10,000 x g for 4
minutes.
2. The pellet is resuspended in a volume equal to the cell pellet of 10
mM Tris hydrochloride (Tris-HCl, pH 8.0) buffer containing 0.1
mg/ml of chloramphenicol and 3 mM NaN3.
3. The cell suspension is centrifuged again at the same settings.
Discard the supernatant.
4. The cell pellet is suspended in an equal volume of spheroplasting
buffer, containing 20% sucrose, 30 mM Tris-HCI (pH 8.0), 0.1 mg
of chloramphenicol per ml, and 3 mM NaN3.
5. The cells are then converted to spheroplasts by incubation at 0°C
for 30 min in the presence of 1/10 the cell pellet volume of 1mg/ml
lysozyme freshly dissolved in 0.1 M EDTA (pH 7.5).
6. Spheroplast and periplasm were separated by centrifugation at
10,000 x g for 10 min.
7. The supernatant contains proteins that are secreted into the
periplasm. This is a convenient method of periplasmic extraction.
Protocol taken from
Ueguchi, C. and K. Ito (1990). "Escherichia coli sec mutants accumulate a processed immature
form of maltose-binding protein (MBP), a late-phase intermediate in MBP export." J Bacteriol
172(10): 5643-9.