Section 7
Preparation of
E. coli
Lysate
For
E. coli
cultures expressing medium to high levels of
Profinity eXact-tagged proteins, (
≥
10% of total protein),
50 ml of culture will yield sufficient material for a 1 ml
cartridge purification, and 250 ml of culture will yield
sufficient material for a 5 ml cartridge purification run.
For cultures expressing protein at low levels (
£
10% of
total protein), the culture volumes will need to be
determined empirically for each protein.
Lysate Preparation
1.
Harvest cells by centrifugation.
2.
Determine weight of cell pellet and resuspend in
10 volumes of bind/wash buffer (50 ml of culture
typically yields 0.5 g of paste, resulting in
approximately 5 ml of lysate).
3.
Sonicate the lysate (on ice) for 3 minutes.
4.
Centrifuge the lysate at
≥
16,000 x g for 30 min at
4°C.
19
Fig. 5. Partioning profile.
Precision Plus Protein™ molecular
weight markers were loaded in lane 1, followed by the total, soluble,
and insoluble fractions in lanes 2–4 respectively. The gel depicts
Profinity eXact-tagged Maltose Binding Protein, which partitions
into the soluble fraction and can be purified using the native
purification protocol. A representative chromatogram and gel for
the purification of this target protein is shown in Fig. 6.
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