1. Harvest the bacterial cells by centrifugation at 3000g for 15 min at 4°C.
   15 m

2. Wash the cells with bacterial cell lysis buffer to remove the residual culture medium. Harvest the washed cells by centrifugation at 3000g for 15 min at 4°C.

3. Decant the supernatant. Weigh the wet pellet.

4. Resuspend the washed E. coli cells in ~3mL of lysis buffer per gram of cell pellet. Stir the suspension for 30 min at 4°C.

If the pellet is not fully resuspended after 30 min, mix the suspension in a Waring Blendor at low speed for ~1 min.

5. Add lysozyme to a concentration of 0.1% (w/v). Incubate for 35 min at 4°C, shaking gently.

A faster rate of lysis can be obtained by increasing the lysozyme concentration to 1.0% (w/v) (i.e., 10mg/mL). Under these conditions, satisfactory lysis can be accomplished in as little as 5 min at temperatures as low as 4°C (for information on protein lysis, see Bollag et al. 1996).

6. Add in the following order (final concentration given):

NP-40 0.5% (v/v)

MgCl₂ 5mM

DNase I 40µg/mL

7. Stir the suspension for 30 min at 4°C to remove the viscous nucleic acid.

Bacterial extracts are 40%-70% protein, 10%-30% nucleic acid, 2%-10% polysaccharide, and 10%-15% lipid (Worrall 1996). The release of DNA upon cell lysis often results in a highly viscous extract that can cause serious problems in subsequent chromatographic purification steps. In addition to DNase I treatment, DNA can be removed from the cell extract (along with other nucleic acids, and in some cases, highly acidic proteins) by adding a neutralized solution of positively charged compounds such as protamine sulfate (up to 5mg/g wet weight of cell pellet) (see, e.g., Scopes 1994) or polyethyleneimine (Burgess and Jendrisak 1975). Methods for DNA removal involving positively charged compounds should not be used with inclusion body preparations, because the precipitated DNA will cocentrifuge with the inclusion bodies.

8. Centrifuge the suspension at 23,000g for 30 min at 4°C. Reserve the supernatant.

9. Resuspend a small portion of the pellet in 1X Laemmli sample buffer containing 100mM DTT.

10. Analyze aliquots of both the soluble protein fraction (i.e., the supernatant from Step 8) and pellet fraction (from Step 9) for the presence of the target protein using analytical SDS-PAGE.

If the bulk of the target protein is found in the insoluble pellet fraction, then inclusion bodies have likely formed, and the target protein will need to be solubilized and purified. If the target protein is found in the supernatant, this material should be stored at 4°C in readiness for the next purification protocol(s).