I'm expressing a mammalian protein in E.coli. My problem is that although protein is expressing (checked by Western blot), its not binding to the Ni-NTA resin (protein is His-tagged). I tried purifying in denaturing conditions, I tried changing the His tag to N-terminal and C-terminal both, and GST tag but the protein binds weakly and elutes in wash. I tried a shallow imidazole gradient and a pH gradient also for purification. I'm getting no protein at all. Please suggest how can I overcome this problem?
Have you verified by western that your protein also has the His tag? If not, I would restain your western using an anti-His antibody. If you already ran a co-immunostain, can you be more detailed about your purification steps? What is the column running buffer your protein is in? Is it not binding at all to the Ni column, or is it coming out in the wash?
was GST construct also tried in denaturing condition. if not, hint is that tag size has to be increased.
Hi,
Has some other laboratory purified this protein earlier? If you do not see an expressed band on coomassie staining, you are in trouble. You can try a host of conditions varying IPTG, temperature, cell density, etc to get enough protein expressed. Trouble shooting purification is easier if you see the protein on SDS-PAGE. Caution:for some proteins E. coli expression for large scale studies just does not work.
Hii everyone..Daniel, I used a protein-specific antibody (commercial) for the western. I'll do the western with the His-tagged antibody now. I'm using 50 mM phosphate buffer pH 7.4, NaCl 150 mM, 5% glycerol. The protein binds to the resin weakly and elutes in 20 mM imidazole alongwith many contaminating bands.
Ashish Sir, denaturing conditions were not tried with GST construct. I thought GST is a good soluble tag to pull my desired protein alongside. My protein is a filamentous protein. Could that be, in any way, detrimental to folding?
Sujay, this protein has been purified by many labs and the crystal structure is also available. Unfortunately, none of their purification conditions have worked for me. The expression level is very low and cannot be seen by Coomassie stain. I tried everything (IPTG, temp, etc) but the expression doesn't increase. It would be great if you could give me some pointers on increasing expression.
Hi,
Was the crystal structure solved with E. coli expressed protein? If yes, then follow that protocol and try variations around it. Is there some activity in this protein that you can check: enzymatic or binding to some ligands? There is a limitless number of things that one can try (codon optimization, different strains of E. coli, co-expression with chaparones, etc). Unless you have a robust and a quick read out for expression (functional?) these experiments will only add up in the frustration column. I have been through this and can vouch for this. Write to the lab that did the structure and they may give you specific pointers. If the protein is important to their future, they may not give you a straight answer.
Good Luck
Sounds like you have a number of problems. First you need to verify your his tag is actually attached to your protein and that it has multiple his residues. Remember that the number of histidine's are related to the strength of your binding. When you run the his western your protein should be highly expressive of histidine (I have a feeling that it is not). Second you are working with a filament which means that the tertiary structure is likely collapsed and possibly masking your his tags (So to answer your question to Dr. Ashish, yes your folding structure is likely compromised). Third, be aware that many filaments tend to form heterogenous structures, so what exactly are your contaminates? (They may just be different varieties of your protein). Be aware filaments are easy to make but difficult to keep stable once the cell is lysed.
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