Hello,
I was working to purify GST-tagged proteins expressed in BL21-DE3 strains. I was having some trouble with the formation of viscous stuff following sonication for the first couple of experiments. So I have added 5 units of DNAseI per mL and 5mM MgCl2 which reduces the viscous stuff but not all. I ran SDS-PAGE gel a couple of times, I found a nice band of GST while GST-tagged protein was way low. We think that it could be possibly due to the less solubilization of my GST-tagged protein. Can anyone suggest to me how to troubleshoot this? Any suggestions will be appreciated!
I'm attaching my lysis protocol below along with a gel image.
1. Grow shaking at 37 C to OD600= 0.6
2. induce with 0.1 mM of IPTG and grow the samples for 3 hrs in a 37C shaker
3. Spin down at 7,000g for 7 min at 4C
4. resuspend the pellet in 10 ml of pre-chilled STE Buffer (100 mM NaCl, 10 mM Tris, 1 mM EDTA, and dd-water) with 1 cOmplete mini protease inhibitor tablet. Spin-down at 7,000g for 7 min at 4C. Store at -80C
5. pellet on ice for about 15 minutes. resuspend in 10 ml cold STE buffer containing protease inhibitors and 100 ug/ml of lysozyme
6. Add 5uL/mL of DNAse I and 5mM MgCl2. Mix and place the tube on ice for 15 minutes
7. after 15 minutes add 5 mM DTT and 1.5% Sarkosyl (10% stock solution).
8. Sonicate cells to lyse (
For GST, Sonicator set at 40% duty cycle for 30 sec, 60 sec off; 2X 90% duty cycle for 30 sec, 60 sec off in between.
GST-protein, sonicator 50% duty cycle for 30 sec, 60 sec off; sonicator 20% duty cycle for 30 sec
9. Add cold 1% Triton X-100. spin down for 10 min at 4C at 8000g
Thank you,
Samina.
Observed viscosity must be caused by genetic material in lysate which solubility improvement has been seen after adding DNase. If the GST-tagged protein is a kind of membrane protein (or has similar hydrophobicity) it forms aggregates rather than viscosity and this is induced by heat treatment. Increase Sarkosyl and/or Triton concentration or try to add SDS to promote solubility (RIPA e.g.). On the other hand, the tagging process may not be successful. Recorded MWs of the GST and POI and tagged POI already clarifies the issue as you indicated. GST cannot be removed without special proteases such as thrombin, factor Xa, or PreScission protease. Therefore it is better to go back to the vector cloning step.
Additionally, did you perform GST-tag affinity purification after lysis or did you conduct any immunoblotting experiment to confirm versatile/efficient tagging?
In a previous job of mine, with GST tagged proteins we were having a problem with getting lots of free GST and only small proportions of the tagged protein that we knew was present based on whole cell lysates (leading to most of the protein not being the final purified product). We eventually worked out that the problem was with a metalloprotease cleaving the tag in the cells, as the problem was drastically reduced by adding 1 mM EDTA and 1 mM EGTA (we were using EDTA free protease inhibitors), with the cleavage effect being further reduced as the concentration of EGTA was raised. We assumed that the metalloprotease must act on the linker to the GST as some position.
Your gel isn't labelled, so I'm unsure what each lane represents, but from your description (are you purifying the protein by the way?) and what I assume is in each lane, I'm not sure if solubility is the issue (the sonication along with the DNAse I should have denatured any DNA). It does sound like a very similar case to what I described above, so try adding 1-2 mM EGTA to all of your buffers in addition to the EDTA.
Dear Samina
probably i'm wrong but the fact that you have many GST alone and few fusion in my opinion can be related to degradation of your gst-protein fusion at the linker region be proteases than insolubility of your protein since in this case you will observe the presence of all the insolubile fusion in inclusion biodies.
i think that you can try to change your expeession condition be decrease the induction temperature. you can try 5h at 25°C or O/n at 20°C and see if in this case the fraction of intact gst-protein fusion is increase since wt lower temperature also the protease acticity will be lower.
good luck
Manuele
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