When ever people make a clone with His Tag to their protein for purification by affinity chromatography why they mostly use only 6 His to protein. What will happened if we put more His suppose 8 or 10. I think by increase no. of His we can increase affinity of our His tagged protein hence chances of getting pure protein will be increased because it will elute with higher concentration of Imidazole.
His6 tags are encoded in old generation plasmids which are still in use in most labs.
Newer vectors encode for up to His10 tags which increase the affinity for IMAC columns as you expect.
There are caveats to using larger tags, however: long sequences are more flexible, longer tags might reach specific or aspecific binding sites leading to aggregation, higher amounts of imidazole might increase the probability of degradation or destabilize the eluted protein.
All the above need to be considered to tailor your construct to the level of purity and properties of the protein sample you aim at. A good approach would be to clone your protein sequence into multiple vectors using ligation independent strategies.
His6 tags are encoded in old generation plasmids which are still in use in most labs.
Newer vectors encode for up to His10 tags which increase the affinity for IMAC columns as you expect.
There are caveats to using larger tags, however: long sequences are more flexible, longer tags might reach specific or aspecific binding sites leading to aggregation, higher amounts of imidazole might increase the probability of degradation or destabilize the eluted protein.
All the above need to be considered to tailor your construct to the level of purity and properties of the protein sample you aim at. A good approach would be to clone your protein sequence into multiple vectors using ligation independent strategies.
Higher imidazole is not good for proteins. It destabilize the protein stability in many case and if u want to put more no of His tag, you can put. but optimize value is 6, as we are following old conception.
Hi Mohd, I believe that the hexa-his tag was chosen because it contains the maximal number of imidazole residues that can coordinate with metal ions.
Increasing the number of imidazoles will not improve binding to metal ions, because all of the coordination sites are already occupied by the 6-His.
As explained by Marco above, the reasons for using His(6) tags are probably historical. Early papers (e.g. doi:10.1038/nbt1188-1321) used up to 6 residues, so -I'm guessing- most people chose that as the 'safe' maximum. Under the right conditions, even a tag with only 2 or 3 contiguous His residues will do the trick.
@Steingrimur: The Ni-NTA complex has only 2 free coordination sites (3 in the case of Ni-IDA), so it can bind a maximum of 2 or 3 His residues, respectively. But even if it had 6 coordination sites, wouldn't in theory a His10 tag bind more tightly simply by decreasing the apparent rate of dissociation of the complex?
Hi Alejandro, you are right, the reason for using 6-His was probably achieved through trial and error, but the Ni-NDA resin can bind Ni++ through 4 coordination sites:
http://www.lifetechnologies.com/us/en/home/references/protocols/proteins-expression-isolation-and-analysis/protein-purification-protocol/purification-of-polyhistidine-containing-recombinant-proteins-with-ni-nta-purification-system.html
Increasing the number of His might increase the affinity, but there is a downside to this:
"Whereas tags of six histidine residues are generally long enough to yield high-affinity interactions with the matrix, both shorter and longer affinity tags have been used successfully. In some cases the use of longer polyhistidine tags has resulted in increased purity due to the ability to use more stringent washing steps.11 Still, it is advisable to use the smallest number of histidine residues as required for efficient purification to minimize possible perturbation of protein function. In general, a six histidine tag is an appropriate choice for the first trial when adding a novel polyhistidine tag to a protein."
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909483/
@Steingrimur: You probably read my post too fast, as we both agree on that particular point. Yes, NTA binds Ni++ through 4 coordination sites. That leaves only 2 FREE coordination sites for interaction with His residues.
I guess another issue to consider is whether you are planning to remove the tag (that is use expression vectors with cleavable His-tags) or not and what you are planning to do with the protein after it is purified.
As Steingrimur mentioned, sometimes longer tags can negatively influence protein folding and/or function so if you are not cleaving the tag it may be better to go with the shortest one.
To follow up on what I stated before, and as Dolors pointed out, high imidazole concentration in the elution buffers can be avoided. There are two other ways of detaching His-tagged proteins from IMAC columns: (1) lower the pH below 6.5 (2) add EDTA or other metal chelators.
In both cases no imidazole is required and, especially method (2) the protein environment is preserved, causing less "stress" to delicate proteins. In case (1) however, the technique is not advisable if the protein pI is around 7.0. It is essential to keep your working pH at least 1 unit (better if 2) above or below the protein pI.
HTH
If the point of cloning and expressing a protein is to characterize the protein (e.g. enzyme activity and its regulation), then even the presence of six histidine residues may change the properties of the protein. I assume the compromise is accepted when the protein cannot be purified directly from the tissue; the alternative being no information at all. In that context, I think keeping the number of histidine residues as low as possible is advisable.
I don't know what I am doing in this distinguished forum, but adding more hisses suggests to me that you might have difficulties eluting your his-8 or -10 tagged protein from a column. Moderation in all things, said Aristotle.
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