I have cloned a gene in Suffle T7 express strain using pET28a vector for expression and in vitro studies. After Ni-NTA column purification of this His Tagged protein, I am getting multiple bands in SDS-PAGE gel instead of one.
What could be possible reasons?
Hello Afshin,
As a first measure, I suggest that you try a more stringent washing before eluting the protein from the Ni-NTA resin. 25 mM imidazole pH 8 won't elute most His-tagged proteins but will get rid of most non-specific binding proteins. If you suspect that your protein forms complexes with other E. coli proteins, you can also wash the resin with high salt buffer. Binding to Ni-NTA is not affected by NaCl concentrations up to 4 M.
The band below the genuine species may be a His-tagged fragment due to proteolytic degradation. To get rid of it, you will need to use other techniques such as gel filtration
The are a number of possibilities here. Firstly, these may simply be contaminating proteins that can be separated by optimising your IMAC experiment. You can do this by introducing more stringent washes to your run as already explained above by PB.
The second possibility is that your protein may be forming higher order oligomeric states that also bind and elute from your IMAC run. This would explain the larger-sized "Non-specific bands". However, these would have to be very stable oligomers to survive the highly denaturing conditions typical of SDS-PAGE.
Lastly, the small band might be due to degradation of your protein in the host or during purification. Add a protease inhibitor cocktail would be a good idea to test this.
Do you have an anti-target protein antibody, or anti-His antibody? A Western Blot would help exclude or confirm some of these possibilities. Alternatively, peptide mass fingerprint or MS/MS sequencing mass spec would also assist.
Unless the level of expression of your protein is very high, it is usually not possible to purify it to homogeneity solely by using IMAC. You should add a second chromatography step, such as gel filtration or ion exchange to improve the purity.
IMAC columns tend to bind a number of well-known E. coli contaminants (proteins with His-rich surface patches, etc). You might try transforming the construct into something like the NiCo(DE3) strain from NEB, where these proteins have been modified through the addition of a chitin-binding domain, doing the normal culture/IMAC purification run, and passing the resulting preparation through a chitin column.
Another thing to try would be IMAC in the presence of a chaotrope (8 M urea or 6 M GuHCl, for instance). If these contaminants are proteins interacting with your protein, the chaotrope should abolish those interactions but not those of the His(6) tag in the target protein with the immobilized ion.
Also, you should try doing a Western blot of those IMAC fractions, just to rule out that these are not proteolytic degradation products or multimers.
And by the way, how high is the level of overexpression of the target protein in the crude lysate? While Adam's comment is true, it is surprising that the relative amount of contaminant species is similar to that of the target protein. If the proteins is being expressed only at low levels, it might pay to focus on increasing the level of expression rather than tuning the downstream purification steps.
Hope it helps!
1. As mentioned above, because the Ni-NTA resin may bind some proteins non-specifically, it is normal to have many protein bands only after affinity chromatography. You need to purify the target protein through further operations, such as gel filtration or ion exchange;
2. If these bands are the degradation bands of your target protein, suggested that the conditions of expression and purification are not sufficient to ensure that your protein is in a relatively stable state, and it is recommended to optimize the conditions, e.g. try more constructions and mammalian cell expression system…;
3. If these bands are endogenous and of high concentration, it is recommended that you optimize expression conditions or try other expression systems.
Hope it helps!
- Badges
- Science method