If you want to screen for expression in small samples, resuspend your cells in buffer (e.g. PBS, Tris-HCl etc.) + 25 u/ml Benzonase. Benzonase is a protease-free DNAse that helps to fluidify the lysate. Add 1/10 vol of lysozyme 10 mg/ml and incubate at 37 °C for 10 min, followed by 1/20 vol Triton X-100 20 % at 37 °C for 5 min. Triton will fully lyse the cells, but will not solubilize insoluble recombinant proteins present in inclusion bodies. Thus, you can centrifuge your lysate, dissolve the pellet in hot SDS-containing sample buffer and analyze it by SDS-PAGE together with the supernatant to assess whether your protein is produced in soluble or insoluble form

Using lysozyme is a fairly cost effective way to do this. I first make up a buffer (50mM Tris.HCl pH 8, 100mM NaCl and 1mM EDTA), then add 20ul of 10mg/ml lysozyme for every ml of buffer. I usually incubate the resuspended bacteria in this buffer at 4oC for 1.5h and it should become viscous. Good luck!

French press like homogenizers and bead beating in an appropriate buffersystem (depending on protein and perhaps next step in purification sequence) can make you avoid detergents and lysozyme altogether. Protease inhibitors and benzonase though will often incease you yield significantly. . . ofc you need the equipment for this, if it is not available and you are not inclined to do the investment, the lysozymes protocol described above is the way to go.

I have sonicated E. coli as follow with excellent results. I collect the cells by centrifugation, wash them with 50 mM Tris–HCl buffer (pH 8.0) + 1 mM PMSF (protease inhibitor) and sonicate in ice by ten cycles of 30 second bursts with 30 second cooling intervals (amplitude 90%, cycle 0.5 in a Branson Sonifier 450 W ). The resulting crude extracts are centrifuged at 20 000 rpm for 20 min at 4ºC to remove cell debris.

I have successfully used french press (3 times) with a 20 mM Tris pH 7,5; 500 mM KCl, 20 mM Imidazol, 10% Glycerin + 2mM PMSF & 1/10 vol of lysozyme 10 mg/ml (30' incubation and rotation at 4°C before french pressing) After using the french press you of course have to centrifuge to get rid of cell debris.

I think the buffer condition you have to test and adapt for you protein.

The following method is about as cheap as they come and doesn't involve the use of mechanical disruption (e.g. expensive equipment) or even high speed centrifugation. It relies on liquid Nitrogen for lysis and produces great results.

The following buffers are used:

2X Lysis Buffer (100 mM Tris-HCl pH 8.0, 1 M NaCl, 60 mM Imidazole pH 8.0)

10X Lysis Buffer (100 mM Imidazole pH 8.0, 20 mM MgSO4, 10% v/v Triton X-100, 5% w/v Na. deoxycholate)

Protocol begins after collecting cell pellet and volumes are designed for 1 L culture (scale accordingly for smaller volumes).

Resuspend cells in 10 ml of 2X Lysis Buffer + 30 ml of ddH2O.

Add lysosome to a final concentration of 10 ug/ml. Incubate RT for at least 10 minutes.

Set up beaker (~400ml) inside styrofoam cooler containing liquid Nitrogen (LN2). Add LN2 to beaker (~halfway).

Add resuspension dropwise to beaker (use serological or pipet). Make sure the tip of pipet is above the top of cooler. 10 ml pipet works best. LN2 will evaporate from the beaker while adding resuspension, so make sure you are filling the beaker with LN2 between additions if it gets too low. Ideally you want small beads (try to keep them in the beaker).

Remove beaker from cooler and allow LN2 to evaporate. Swirling in a 37°C water bath helps speed up the process.

Add 30 ml of 2X Lysis Buffer plus 10 ml of 10X Lysis Buffer. Add 2ul Benzonase (or cheaper DNAse like SuperNuclease). Incubate at RT for 15 min.

Apply to Ni-IMAC column (or method of choice for 1st purification step, I prefer to use gravity flow columns).

Dear All,

Thank you for the suggestions provided.

Regards

Santosh

Source of another cheap nuclease is here: http://www.protean.cz/en/recombinant-protein/11/endonuclease-s-marcescens