Hello Dear friends, I have one of my protein which I’m trying to purify it by His-affinity chromatography put after lysis the bacteria cells pellet with lysozyme and centrifuge it, most the protein still in the pellet and I didn’t get enough protein after purification, that happened maybe the bacteria forming the inclusion bodies due to overexpress so, I tried to reduce the IPTG concentration and the time .of incubation, but the problem stilled, I used some iconic detergent like 1% Triton, and I couldn't make any difference.
If someone has any idea or suggestion, it will be awesome
Hi Ali
Please have a look on this homepage. They explain it verry well how to extract the proteins.
https://www.thermofisher.com/sg/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/his-tagged-proteins-production-purification.html
Good luck
Hi Ali
Add to the pellet 6-8 M urea or 6M Guanidine hydrochloride. This is compatible with some purification protocols like IMAC, SEC.
Using strong denaturants should be the last option; refolding proteins can be a pain in the ass.
If you are working with the membrane protein you can extract it from the membrane by 3 hours incubation at RT with detergent (ex. 20mM DM in the presence of 5mM Imidazole and 150mM PMSF). In order to reduce cysteine residues you can add 5mM BME to the solution. Then you spin it down with the ultra speed (ex. 37 000 rpm at 4C) and save a pellet for the purification.
3 Methods here to bypass inclusion your protein going into inclusion bodies:
Method 1) Don't use bacteria.
I am not an expert at this but I read up new techniques quite frequently.
One thing that you could probably do is to solubilize the protein by in vitro transcription and translation of your protein using Wheat Germ Cell Free protein synthesis. I was wondering where the ribosomes and important stuff for translation was but it's all in the wheat germ embryos.
If you do this then there will be no inclusion body formation because there is no bacteria present. I can't tell you how much protein you could expect to get, but this procedure has been used for cool things like unnatural amino acid incorporation to proteins.
See Chapter 4 of this book (attached) Methods in Molecular Biology, Volume 1621 Plant Receptor Kinases Methods and Protocols for a very detailed procedure.
Method 2: Add an affinity tag that is soluble
Another thing that you could do is add a very soluble tag such as the SUMO tag to your protein, instead of multi-histidine tag, (using fusion PCR?). This tag should make your recombinant protein soluble and thus take it out of the inclusion body altogether. Then the SUMO tag can be very specifically cleaved and you get your protein. This might be useful:
Article SUMO as a solubility tag and in vivo cleavage of SUMO fusion...
Method 3) Consider a new E. coli expression strain
Maybe there is some correlation between inclusion body formation and the AT or CG richness of the gene that codes for your protein and your choice of E. coli expression strain. So try a new strain of E. coli that isn't likely to put your protein into an inclusion body.
Personally I think you should use Wheat Germ Cell Free Synthesis of your protein, but the SUMO solubility tag would probably be very effective, too!
Hope this helps!
Omar Gonzalez-Ortega
Thanks for your replay, I have been already reduced
Dear Oscar Otero Alfaro I need the protein for the enzyme activity, so this option it will not helpe me for that.
Thanks a lot for your suggestion
Dear Julia Skalska this protocol during the induction experiment ?
and these detergent might will effect for the activity of my protein, which I need it for enzyme activity.
I`m waiting for your answer please
with regards
Dear @ Murtadha Ali this is the protocol after you finish oveexpression of your protein and extract it from the bacterial pellet. I used it for the membrane proteins, precisely ion channels. The detergent which I used is only an example and probably you will want to do your own screening and optimize the conditions for your protein. I hope it will help.
Good luck!
Murtadha Ali , one more thing. You could try various concentrations of Lactose Induction of the Lac Operon, instead of IPTG induction of the lac operon. IPTG is toxic to cells as it is a non-metabolizable analogue of lactose which binds irreversibly to the lac operon, rapidly producing protein, sometimes so fast that the protein does not have time to properly fold when it is being expressed. Lactose suffers from none of these complications, but when doing lactose induction the lac operon is inhibited by glucose in the media, but apparently not too much at low concentrations of glucose this is not a problem.
Finally, if your protein is a membrane protein you just have to live with your problems because membrane proteins have a transmembrane region which means hydrophobicity. I tend to think that also if you reduce the rate (rpm) at which you centrifuge then less proteins should fall into the pellet based on how centrifugation works.
Alos, a method to obtain the most viable cells is to do double colony selection on X-GAL plates for blue-white screening, supplemented with your appropriate antibiotic .
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