his tag purification is quite common this days. One little problem are those proteins thas naturally have a polyhis in their sequence and that will tend to copurify. My question is if someone knows which are the most frequent contaminants of an his tagged recombinant protein purified from saccharomyces cerevisiae.
According to me, because it is affinity chromatography, the contaminant during his-tag protein purification could be only the other undesired his tag containing proteins for any model. And I guess imidazole ring has very high affinity for the nickel metal ion.
This is the just a logical attempt, correct me if I am wrong.
Thank you,
Rakesh.
Probably the easiest way to determine this definitively would be to perform a Protein ID mass spec analysis on a dummy purification where (under given conditions of pH, salt and imidazole concentration) straight E.coli lysate is passed over NiNTA resin, and the composition of flow-through, various washes and elutions is compared. It would comprise a really nice study if it could be funded somehow - also comparing different IMAC resins and conditions to minimise significant contaminants in the absence of an expressed protein, and then comparing these when a defined His-tag protein is included. This could also allow for comparisons of the effectiveness of differing variants of His tags and resins for purification.
With many proteins (not just His-tagged), significant contamination from E.coli can comprise various chaperone proteins that are invoked by the unfolded protein response in bacterial cells (GroEL, DnaK, Hsp70 etc.). These can be extremely difficult to remove. There are also cytochrome proteins that bind to Nickel resin (giving the characteristic yellow tinge that elutes between 10-50mM Imidazole at pH8).
See also Carine Robichon's excellent paper from 2011 :
Appl Environ Microbiol. 2011 Jul; 77(13): 4634–4646.
doi: 10.1128/AEM.00119-11
PMCID: PMC3127686
Engineering Escherichia coli BL21(DE3) Derivative Strains To Minimize E. coli Protein Contamination after Purification by Immobilized Metal Affinity Chromatography ▿ † ‡
Carine Robichon, Jianying Luo, Thomas B. Causey, Jack S. Benner, and James C. Samuelson*
Good luck!
Hi, thaks for the answers. The problematic co/purifying proteins do have 6xHis or even more ( I found one with a run of 13 xHis) in their sequences. A study of this contaminating proteins has been done in E.coli but we are working with saccharomyces cerevisiae. I think the study proposed by Robin is quite appropriate but I wonder if no one has done it yet with yeast his tag purified proteins.
In my experience it depends a lot on supplier of the column material. We routinely see that suppliers A's columns co-purify the same contaminants in every run but that these are less present or absent in supplier B's material... and vice versa... also it depends of cause on the specific experimental setup (buffer, pH, ionic stength, elution profile, etc.) but even when these are similar, scaling i.e. beads per protein can affect the output in specific amount of contaminant. Also note that not all suppliers have similar bead quality regarding polymerization of bead material, bead uniformity, etc.
I know for a fact that some metal transporting ATPases in yeast have long intrinsic His stretches so these could well turn up to be contaminants.
hope this helps...
Thank you Danny, your suggestion of comparing different columns is a good thing to try.
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