I am trying to purify membrane bound beta-lactamase protein in BL21 E.coli strain. I have created soluble construct of the gene by removing the signal sequence from 5' and 45 nucleotides removed from 3' end to minimize the membrane anchorage at carboxyl terminal of the protein for pET28a(+) vector. After induction with IPTG (1mM), incubation is done for 3-5 hours at 37 degree celsius. I tried lysing the cells with lysis buffer and sonication separately. Under sonication I am getting the protein in cell pellet while lysis buffer yield protein in supernatant as well as pellet while the combination of both yielded protein in pellet.
Please help me in getting the protein in supernatant fraction for further purification process.
I guess that sonication. Beware to submerge the tip in order to not form foam and use cycles of sonication and rest (4 cycles, 15-20s of each should be enough) leaving the sample on ice while resting so it doesn’t heat. Foam and heat will denature your protein which produces aggregates and precipitation.
I've had better success and better quality protein when I use detergent based lysis compared to sonication. I flash freeze my pellets in liquid nitrogen immediately after harvesting then put them into 200mL of BPER (G-bio 786-177) with protease inhition tablets (EDTA free for his-based purification), DNAse, RNAse and lysozyme. I add a stir bar and stir for ~4 hr in a cold room. Hope this helps!
I would add that if you decide to use a detergent based lysis solution, some are much stronger than others. Please be aware. I have used a Triton-X based lysis solution (plus protease inhibitors, etc), that has worked really well for me in the past. I also used a lysis solution with low concentrations of Triton-X, SDS, and Digitonin, and it was clearly too strong for my needs, as is lysed the nuclear envelope, and I ended up with DNA smears in my western blot. I don't remember precisely what concentration of Triton-X I used, but it was really low.
Katie; This is one of the big challenge we all have. First you decide what purpose you are preparing membrane protein is most important. Since you are trying to prepare from E.Coli cells and not interested in studying any interacting partners suggests you can use sonication with mild pulses (10-20 psi for 5s, repeat five times with 30s intervals) with in the ice. Your buffer should have protease and phosphatase inhibitors which avoid proteolysis. After sonication, add non-ionic detergents like TX-100 up to 2% (that is more than enough) incubate at cold room for 30 minutes with end-to-end mixing. I hope you will be able to get your protein almost 90% in the sup as a soluble form. In case, if you do with tissues or isolating interacting partners with your interested protein, sonication is not recommended and prefer to use lysis method. To avoid DNA smears, add few microliters benzonase during incubation.
Hope this will work for you. All the best.
Saro.
You could try the combination of both, but insted sonicate the sample with a needle type device, try a bath sonicator. We get good results sonicating samples pre-treated with a lysis buffer for 30 min.
Hope you find this helpfull.
All the best.
Hi Gaurav:
You should try RIPA Buffer in order to get a soluble fraction of your recombinant protein. I am pretty sure it will work.
http://www.sigmaaldrich.com/life-science/proteomics/recombinant-protein-expression/cell-lysis/mammalian-cell-lysis/ripa-buffer.html
Good luck!
Thank you very much for your valuable suggestions.
@ Rimma- yeah, my next step is to concentrate the protein with Ni-NTA.
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