I am working on the transcription factor. I am trying to get protein and for that, I did molecular cloning of gene into e.coli. But I am facing a problem that protein is going into the pellet. I used pET28c and pET28b vectors both, protein is having c ter his tag. In both cases, it is not soluble. I harvested the pellet after expression (16*C and 37*C, induced the culture at 0.6 OD, and kept for overnight and 4 hrs respectively). I performed lysis of pellet by sonication (30% amp, 10 sec on & 10 sec off for 20 minutes). I also used 8M urea in buffer and incubated for 30 minutes and then sonicated it, it is still going into pellet. How to solubilize it? I am attaching the gel picture of it.
FIG:- Consider only the first three wells.
Description of wells-
Lane-1 Culture Pellet + 8M urea incubation 30 min followed by sonication for 15-20 minutes supernatant
Lane-2 Culture Pellet +8M urea incubation 30 min followed by sonication for 15-20 minutes pellet
Lane-3 Culture Pellet +8M urea incubation 30 min followed by sonication for 15-20 minutes (mixed, before centrifuge)
Dear Chankit Giri
Just some questions:
- Since you wrote that your protein is a transcription factor, it is not a membrane protein, right? It do not contain any transmembrane regions?
- Which is the exact composition of your urea buffer? Eg pH? and which is the pI of your protein?
- How much buffer with UREA 8M do you use for each g of the pellet?
As general consideraiton.
To improve your extraction you can try to:
1) reuspend and homogenize well your cells with a pestle (eg https://www.sigmaaldrich.com/IT/en/product/sigma/p7734) before sonication.
2) Use Guanidine-Hcl 6M buffer instead urea buffer.
Pay attention that samples with guanidine need to be diluted before load it in SDS-page (final guanidine concentraion have to be max 1M) otherwise the solution willl precipitate, since guanidine react with SDS.
good luck
Manuele
Manuele Martinelli thanks for the response. Here are the answers of few questions, that you have asked. Thanks for the suggestions also, I will consider them.
-Yes it is a member of GntR family of transcription factors (TFs). No transmembrane domain is there as per PRED-TMR.
Composition I used for basis buffer- 50mM NaCl, 300mM NaH2Po4, 8M urea. pH of the buffer is 8. pI of protein is 5.83.
I generally use 300ml expressed culture pellet suspended into 30 ml of basis buffer, further 5 ml of it I took and mixed with 25 ml buffer (same buffer) and sonicated it.
After sonication, centrifuged at 12000 rpm for 20 minutes and loaded on gel and found that protein is present in pellet, not in the soluble fraction.
I will consider gHcL.
Omar Gonzalez-Ortega you mean Incubation of bacterial pallet in 8 M urea?
Dear Chankit,
You can harvest your cells in a native buffer at a low rpm for 15-20 minutes then incubate with lysozyme for 30 minutes at room temperature, then sonicate your samples for 5 min at 50% amplitude (with on and off cycles), centrifuge and collect the pellet (low rpm for 15-20 min) and add it in a denaturing buffer (native buffer +denaturant (8M urea) and incubate at 37 degrees for 1 hour under shaking conditions and then centrifuge at say 10-12k rpm for 30 min and collect the supernatant. You will have your solubilized protein. Now perform dialysis to refold your protein (preferably step-wise). Good Luck
Roshni Mohan thanks for the suggestion,
I will use this and get back to you .
Dear Chankit,
Please share the protocol you have done. I will suggest if any changes to be done.
Roshni Mohan
I harvested cells in a native buffer at a low rpm for 15 minutes then incubate with lysozyme for 40 minutes at room temperature, then sonicated samples for 10 min at 40% amplitude (with on 10 sec and off 10-sec cycles), centrifuge and collect the pellet (low rpm 9000 for 15 min) and add it in a denaturing buffer (native buffer +denaturant (8M urea) and incubate at 37 degrees for 1 hour and overnight also under shaking conditions and then centrifuge at 12k rpm for 25 min and collect the supernatant.
Protein is still in the pellet.
Hi Chankit could you share the PAGE images and also the composition of the native buffer? Minimize the sonication to five min.
Do you disperse the inclusion bodies pellet after adding urea using the ultrasonication tip?