loss of His tag from protein made in mammalian cells? - Anyone have experience with His tag being cleaved off? (Nov/20/2005 )
Hi,
I have a 105 kDa protein being produced in HEK293 cells (it was toxic to bacteria). It has a 6XHis tag at the C-terminus, and is a soluble protein which can be seen in the cytoplasm, but is not secreted into the cell media.
Immunocytochemistry reveals anti-His labelling which is proportional to the amount of expression of the protein (as evaluated by labelling with an antibody specific to the protein). The HIS signal is weaker than control proteins with HIS-tags, but is present nonetheless.
However, when I harvest cellular proteins and begin to try Ni-NTA purification, there is no HIS signal (by Western) from any of the fractions, including the crude cell lysate. I have tried mild and harshly denaturing conditions, with no luck. It is as though the HIS signal is completely degraded.
I have sequenced the construct, and there are no hidden STOP codons to suggest truncation of the protein. Does anyone have any ideas? Ever experienced spontaneous loss of a HIS-tag?
Many Thanks!
I have a 105 kDa protein being produced in HEK293 cells (it was toxic to bacteria). It has a 6XHis tag at the C-terminus, and is a soluble protein which can be seen in the cytoplasm, but is not secreted into the cell media.
Immunocytochemistry reveals anti-His labelling which is proportional to the amount of expression of the protein (as evaluated by labelling with an antibody specific to the protein). The HIS signal is weaker than control proteins with HIS-tags, but is present nonetheless.
However, when I harvest cellular proteins and begin to try Ni-NTA purification, there is no HIS signal (by Western) from any of the fractions, including the crude cell lysate. I have tried mild and harshly denaturing conditions, with no luck. It is as though the HIS signal is completely degraded.
I have sequenced the construct, and there are no hidden STOP codons to suggest truncation of the protein. Does anyone have any ideas? Ever experienced spontaneous loss of a HIS-tag?
Many Thanks!
I am sorry but Im not familiar with the loss of a his tag but I did notice that you are trying to detect with the his-tag antbody already. Incase its not an issue of loosing the histag:
Have you tried to stain the gel with coomassie blue to visualise the protein in the crude lysate etc?
If you havent this will allow you to visualise your expressed protein (remember to run a negative lystate control to allow comparison). This will verify that you are not loosing your protein due to degradation when you are harvesting your cells (ie need to add protease inhibitors and strictly carry out on ice), or that you are diluting the protein too much in the harvest buffer so perhaps you just need to load more ug lysate to detect your protein. If you can see the protein expressed then your issue may be
- cleavage of the his tag (like you suggested)
- or a problem with your detection of the his tag ie antibody conditions.
Hi,
I neglected to mention before, the Western works fine with a protein-specific antibody (including one which recognizes a region close to the C-terminus, where the HIS tag is). And the protein is the correct size, reinforcing the idea that it is ONLY the HIS tag which is lost.
The HIS antibody works fine, both in Westerns and immmunocyto, with other proteins bearing a HIS tag.
Mammalian cells are uglier than bacteria when it comes to purifying protein: the crude lysate is a giant smear of proteins, and expression of a transgene doesn't really show up too dramatically against this background on a Coomassie gel. However, the ease with which the protein is detected by immunocyto and Western, with specific antibodies, suggests the expression level is adequate.
still stumped........
Does your vector contain enterokinase or thrombin cleavage sites downstream of the 6 x his?
Hi,
I believe I have checked for every cleavage signal.....according to SignalIP 3.0, there are no predicted cleavage signals. Also, no thrombin, enterokinase, or Factor Xa sites in my protein. Any other ways you can think of to purify this thing? I tried cutting a band out of the gel and eluting it with limited success........really need to find a way to concentrate it first and separate it from the chaff of cellular proteins. Is Copper any better than Nickel?
Cheers
I have a 105 kDa protein being produced in HEK293 cells (it was toxic to bacteria). It has a 6XHis tag at the C-terminus, and is a soluble protein which can be seen in the cytoplasm, but is not secreted into the cell media.
Immunocytochemistry reveals anti-His labelling which is proportional to the amount of expression of the protein (as evaluated by labelling with an antibody specific to the protein). The HIS signal is weaker than control proteins with HIS-tags, but is present nonetheless.
However, when I harvest cellular proteins and begin to try Ni-NTA purification, there is no HIS signal (by Western) from any of the fractions, including the crude cell lysate. I have tried mild and harshly denaturing conditions, with no luck. It is as though the HIS signal is completely degraded.
I have sequenced the construct, and there are no hidden STOP codons to suggest truncation of the protein. Does anyone have any ideas? Ever experienced spontaneous loss of a HIS-tag?
Many Thanks!
hi, i have the same problem. 60kD protein his tag on C-terminus. on the western from lysates i can detect the protein with specific antibody, but anti-his gives me no signal at all. however i noticed that on the gel it runs at the size of tagged protein, therefore i simply checked, by doing the IP with NiNTA if the tag is functional. and it works - based on coomasie i can tell, that i pull down his tagged version, but not flag-tagged version, and also vector control is clear.
seems like the tag is working.
however, i have totally no idea why the Ab doesnt recognize it...