HIS-tagged protein purification - (Jun/07/2011 )

I am working on purification of his tagged protein. I am tracking the overexpressed protein by activity. So far the protein goes straight through the Ni2+ column. Does anyone know if adding of polygylcine linker between the protein and the histag would improve binding?

sashacat on Tue Jun 7 14:00:32 2011 said:

Hola as the days run and you haven´t any answer i´ll give you my opinion. For me it´s strange that your protein doesn´t bound having a 6His tag. The colum remains blue after sample pass?. I would revise any of the incompatibilities of the column with sample buffer components before to construct a longer arm. because you could have the same problem,Buena suerte

protolder on Thu Jun 16 06:06:44 2011 said:

The binding buffer is 20 mM HEPES pH=8.0, 500mM NaCl, 10mM imidazol. The column does stay blue after the ran. Given that the protein is monomeric it really puzzles me. I suppose I can try phosphate buffer.

sashacat on Thu Jun 23 13:29:11 2011 said:

Also I tried purifying a different protein at the exact same conditions and it worked well.

sashacat on Thu Jun 23 18:45:27 2011 said:

Try removing the imidazole from your binding buffer - that's all I got. If it still doesn't bind, I'm not convinved there's a his-tag there. Do a anti-His western blot on the crude extract to be sure?

Bluefunk on Sat Jun 25 20:45:43 2011 said:

I did in gel his tag staining using his tag stain from invitrogen and it shows that there is no his tag present. It is odd because I have the activity associated with the enzyme, but not his tag. Can it be that only his tag is being degraded or what else should I try to prevent histag degradation?

So you know your protein of interest is there, but you can detect the his-tag? I'll recommend re-sequencing your expresion clone to confimr the His-tag is actually there, and in frame. I had this problem in the past, was unable to purify my protein through a His column despite being able to detect it on western blot (with protein specific antibodies). When I did an anti-his western I couldnt see anything. I then went back to my sequence,and found out that the His-tag was not in frame and therefore I did not have 6-His at the end of my protein.

Hope this helps.

I had a similar problem with one of my proteins years ago. I don't know which expression vector you are using but the His-tag is usually already in frame with the start codon of the vector so that shouldn't be an issue. With my protein, I concluded that the His-tag was folded somehow inside the protein and was thus not exposed - could not bind to Ni2+ IMAC column or react with anti-His antibody on Western blot. You could try purifying your protein under denaturing conditions which will expose the hidden his-tag for easier purification using IMAC - but then you obviously need to re-fold which will take some time to optimise. Can you use another method of purification such as size-exclusion or the like?

Bea Kerr on Fri Jul 8 06:59:49 2011 said:

The protein is very difficult to purify which is why I decided to go with his tag to begin with. The odd thing is that I attached the his-tag with all the different linkers in between including a long glycine polylinker and it won't bind. Also wouldn't protein be completely unfolded and thus histag exposed when you do SDS PAGE gel before you do Western blot?

thanks for the post. Misery loves company.

sashacat on Tue Jul 12 20:17:30 2011 said:

You are right. On a SDS gel, proteins should be denatured and his tag should be exposed. Are you saying that the his tag is confirmed by DNA sequencing and is in frame with your insert? If that is the case, the only possiblity is that the N terminal of your fusion protein is truncated by the cell. I had one case like that. A c-terminal HIS tag will solve the problem. I suggest you should try our new bioreactors to get higher yield of your proteins so you do not need western to see your protein. Please check out our webpage at qizhonglabs.com for more details.