I'm trying to purify membrane protein from HEK293 cells for x-ray structural determination.
I added the his-tag at the c-terminus of the protein, but the eluted protein from the resin contains huge amount of non specific proteins.
I confirmed that the protein is active.
I asked about his-tag purification on this website before and understood that his-tag is not suitable for purification from human cells.
Now I'm wondering what is the next tag I have to try.
The tag must be small peptide because I don't want to digest the large tag with protease.
Please give me suggestions.
I think, "INTEIN MEDIATED PURIFICATION" may help you in your project.
It is a self-cleaving tag and no need to digest by protease.
The attached link may provide you some information about it.
Good luck
https://www.neb.com/products/e6901-impact-kit
We have used the FLAG tag for 5-HT3R purification.
http://www.ncbi.nlm.nih.gov/pubmed/25460187
In case this might help you - you might wish to also check some of the discussion under the question: "What would you recommend as the best tags for large-scale purification of proteins?"
Hello Kiyohiro,
there are many tags that are suitable for membrane protein purification. We have had good results in our lab with the "1D4" tag (C-terminal TETSQVAPA nonapeptide) and anti-1D4 Ab purification. See e.g.;
http://www.nature.com/nature/journal/v512/n7514/full/nature13293.html
I like to use the N-terminal Twin-strep tag with Strep-tactin resin as well, however this tag would have to be cleaved off;
http://www.iba-lifesciences.com/twin-strep-tag.html
The His-tag is not unsuitable, it is my workhorse tag for purifying soluble proteins from eukaryotic DMEM medium. Stripping of the matrix was always a problem though. But GE now markets a Nickel Sepharose "Excel", that is totally resistant to stripping by small molecule chelators in the medium.
http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences/17371202
You could also use e.g. a deca-His (His10) tag instead of the normal hexa-His (His6) tag. This would allow you to do more stringent washing of the beads before elution. Also, Cobalt resin (e.g. Talon) retains much less contaminants than Nickel resin.
Hope this helps,
Jonathan
If you are using Ni-NTA beads then you can also check with Cobalt resins because cobalt have higher specificity than Ni NTA. So it will specifically binds to your His tagged protein.
Hope this helps,
Good luck
we use (as Jonathan suggested) a double strep for the purification of some of our membrane proteins, however if your SDS PAGE gel is indicating that the protein is very dirty after IMAC then this is down to one of two things either the wash step need optimization or the protein is "sticky" , this can be down to the protein aggregating (check the SEC of the protein post IMAC) and then pulling down a lot of other protein with it typically through hydrophobic interactions, by using different detergents you can get around this but this can also be achieved through stabilization of the protein through optimization of the buffers and additives (such as ligands and lipids) or through mutagenisis. You mention that the protein is active one thing to more carefully look at is the specific activity of the protein, what proportion of the protein is active this is only going to be an estimate as you will only be able to estimate the % of your protein based on your gel, if the % activity is low then it would suggest that the protein is aggregating
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