1. Centrifuge bacteria from
culture (9 Krpm Ja14, 15 minutes). Freeze cell pellet.
2. Re-suspend bacteria in lysis buffer (see below).
3. Add 5mg lysozyme except where its molecular weight (11kDa) is
close to the expressed protein. The
lysozyme helps break cell wall but is not essential.
4. Sonicate with bursts of 1 minute making sure that the
temperature does not increase locally (swirl).
Can use DNAse1 to break up DNA but soncication does a good job./
5. Centrifuge at 12 Krpm (Ja20, 20 minutes).
up the pellet in 50 mls Inclusion body buffer 1 (see below) and stir for
15-30 minutes on bench.
7. Centrifuge at 12 Krpm (Ja20, 20 minutes).
8. Take up pellet in 50 mls Inclusion body buffer 2 (see below)
and stir for 15-30 minutes on bench.
Centrifuge as before and repeat step 8, 3x.
10. Take up the
pellet in 8M urea, 0.1mM NaN3, 1mM EGTA and a suitable buffer (e.g. 10mM
Tris pH8.0) using a hand-held homogeniser.
11. Dilute to 6M urea and centrifuge at 12 Krpm (Ja20, 20 minutes).
12. The supernatant can be applied to a suitable equilibrated ion
exchange column (DE52, CM52). Wash
the column in buffer without urea and elute of the protein with a salt