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Purification of expressed proteins from E. coli inclusion bodies.

This method is for a 1 litre culture of E.coli that results in the accumulation of protein in an insoluble "inclusion body".  This is not necessarily a bad thing as the following protocol results in a fairly rapid purification of protein.  Often the protein can be refolded by this procedure to produce functional proteins.  Crystallographers don't like dealing with proteins derived from inclusions as heterogeneity  making crystals difficult to obtain.  If you want to avoid inclusion bodies, try growing the bacteria at different temperatures and/or in different hosts e.g. BL21 de3, plysS or plysE.  Another trick that sometimes works is to use different media, for example LB, 2xTY or TB.

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).

6.  Take 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.

9.     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 gradient.

This 

Lysis Buffer:-

 

Inclusion Body Buffer 1:-

Inclusion Body Buffer 2:-

50 mM Tris pH 8.0

20 mM Tris pH 8.0

10 mM Tris pH 8.0

1 mM EDTA

0.2M NaCl

0.25% Sodium deoxycholate

25% Sucrose

1% Sodium deoxycholate

1 mM EGTA

 

2 mM EGTA

 

 

 

 

 

 
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