effective chemical lysis of E. coli under anaerobic conditions

From: Jim Spencer, Cellular and Molecular Medicine
Date: 29 November 2011 14:49


Dear All,

Apologies for the off-topic question.  I'm seeking suggestions on the best way to achieve an effective, but not over-harsh, chemical lysis of E coli expressing an oxygen-sensitive Fe-S protein.  We need to lyse anaerobically but do not have access to a sonicator that we can use in the glove box.  We have used a proprietary detergent mix in previous attempts but have not been overly impressed with the results- lots of protein (more than I would expect based on past experiences with related enzymes) remains in the pellet.  I've seen literature protocols based on Triton (up to 1.2%) but am worried (perhaps without basis) that this might interfere with downstream steps (reconstituting the Fe-S cluster and possibly crystallisation).  The protein is His-tagged.

Does anyone know a better way?

Thanks in advance.

Best wishes

Jim

----------------------
Dr. James Spencer,

----------
From: Artem Evdokimov

0.5% octyl-thioglucopyranoside + lysozyme + DNAse in 30 mM TRIS pH 8.0 - degassed/sparged and supplemented with whatever reducing agent you need. Stir in cold for 30-40 minutes at a ratio of 3ml of solution to 1g of cell pellet (wet).

 

Artem