How do histidine tags work?
How do histidine tags work?
A his-tag, or polyhistidine tag, is a string of histidine residues at either the N or C terminus of a recombinant protein. The his-tag has a high affinity for these metal ions and binds strongly to the IMAC column. Most other proteins in the lysate will not bind to the resin, or bind only weakly.
What are two purposes of a polyhistidine tag added to a protein?
One of the most commonly used tags is the polyhistidine tag, also known as His-Tag, which is a string of usually between six and nine histidine residues (see Figure 1 below). This method of tagging is especially useful as it allows for easy purification and detection of the recombinant protein.
How many kDa is a His tag?
2.5 kDa
His-tags, due to their relatively small size (∼2.5 kDa), are not believed to significantly interfere with the function and structure of a majority of proteins.
How does NI-NTA work?
Ni-NTA Agarose is a nickel-charged affinity resin that can be used to purify recombinant proteins containing a polyhistidine (6xHis) sequence. Ni-NTA uses the chelating ligand nitrilotriacetic acid (NTA) coupled to a cross-linked 6% agarose resin that is suitable for use in batch and gravity flow applications.
What functional group of histidine binds to the column?
Histidine (HIS6) Tags The HIS6 binds somewhat specifically to a nickel-nitrilotriacetic acid (NTA) organic functional group that might be bound to an agarose chromatography column, pulling only labeled molecules from the solution phase.
How does histidine act as a buffer?
In a histidine proton shuttle, histidine is used to quickly shuttle protons. It can do this by abstracting a proton with its basic nitrogen to make a positively charged intermediate and then use another molecule, a buffer, to extract the proton from its acidic nitrogen.
How does affinity purification work?
Affinity purification involves the separation of molecules in solution (mobile phase) based on differences in binding interaction with a ligand that is immobilized to a stationary material (solid phase). A support or matrix in affinity purification is any material to which a biospecific ligand is covalently attached.
What is a 6xHN tag?
Distinct types of his tags are available, including the 6xHN tag (12 amino acids) Most common affinity tag used to purify proteins. Binds to coordinated metals such as Ni2+ or Co2+ His-tagged proteins can be eluted with imidazole or low pH buffer. A his tag can be incorporated on either the N- or C-terminus.
Why does His-tag bind nickel?
When a protein having a His-tag is brought into contact with a carrier on which a metal ion such as nickel is immobilized under the condition of pH 8 or higher, the histidine residue chelates the metal ion and binds to the carrier.
Does His-tag need to be removed?
In the vast majority of cases, it is not necessary to remove the polyHis-Tag from recombinant proteins. The polyHis-tag is widely and preferably used for recombinant immunogen production because of its small size (0.84 KDa) and low immunogenicity. Although unusually, His-tag may affect the functionality of the protein.
What is NTA in Ni-NTA?
The term Ni-NTA (Nickel NTA) refers to a nickel2+ ion that has been coupled to Nitrilotriacetic acid (NTA). Ni-NTA can then be coupled to agarose resin or magnetic beads for IMAC (Immobilized Metal Chelate Affinity Chromatography). This is a purification method to obtain functional His-tagged protein.
What is an IMAC column?
Nickel columns are used for immobilized metal affinity chromatography (IMAC) for the purification of recombinant proteins with a polyhistidine tag on either terminus. A recombinant protein with a 6xHis tag has a high affinity for nickel, whereas most other proteins will either bind with low affinity, or not at all.
How many histidine residues are in a suitable tag sequence?
Suitable tag sequences are available free for commercial use; for example, MK (HQ)6 may be used for enhanced expression in E. coli and tag removal. The total number of histidine residues may vary in the tag from as low as two, to as high as 10 or more His residues.
What is Polyhistidine-tagging and how does it work?
Polyhistidine-tagging is the option of choice for purifying recombinant proteins in denaturing conditions because its mode of action is dependent only on the primary structure of proteins.
What is the role of polyhistidine in affinity purification?
Polyhistidine-tags are often used for affinity purification of polyhistidine-tagged recombinant proteins that are expressed in Escherichia coli or other prokaryotic expression systems. The bacterial cells are harvested by centrifugation and the resulting cell pellet can be lysed by physical means or with detergents or enzymes such as lysozyme.
Why do histidines increase the affinity of a protein for metal?
Therefore, if a number of histidines are added to the end of the protein by genetic engineering, the affinity of the protein for the metal ion is remarkably increased and the basic idea is that purification can be easily carried out.
