Poly-his-tag Protein Expression, Production, and Purification Basics

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What is a poly-his-tag?

A polyhistidine-tag is an amino acid motif consisting of six-ten histidine (His) residues, either fused at the N- or C-terminus of a protein. It is also known as hexa histidine-tag, 6xHis-tag, His6 tag.

Why use a poly-his-tag for protein expression and production?

Adding a poly-his-tag to the N- or C-terminus of a protein allows rapid, simple, and efficient purification of the over-expressed recombinant protein, by using either Immobilized metal (Nickel or Copper) ion affinity chromatography (IMAC). In addition, antibodies specific to the poly-his-tag can be used to detect the tagged protein expression level in cell culture, by ELISA and western blot.

How many histidines should I add for protein expression and production?

The number of histidine residues in a poly-his-tag can affect the binding of the tagged protein to the affnity resin and elution of the protein off the affinity column. Usually, a hexa-his-tag is sufficient for affinity purification and antibody detection. However, sometime, 7-10-histidines are used to increase binding of the his-tagged protein to the affinity column, possibily due to unexposure of one or more histidine residues to the resin.

How do I cleave of the poly-his-tag after purification?

In some applications, it is desirable to remove the his-tag, for example, for protein crystalization. To allow cleavage of the tag, a protease cleavage site needs to be engineered between the tag and the protein. An EK cleavage site behind the His-tag (poly-his-EK site-protein structure design) can allow complete removal of the tag and the cleavage site, leaving no additional amino acids after the specific cleavage of the tag, resulting in a native form of the protein. For more information on the cleavage site and tag removal of EK and HRV-3C, please refer to: Enterokinase (EK), HRV-3C (human rhinovirus protease).

How to Purify Poly-his-tagged Proteins?

Poly-histidine-tag binds to bivalent nickel or cobalt ions chelated by iminodiacetic acid (Ni-IDA) and nitrilotriacetic acid (Ni-NTA) on sepharose resin/agarose, which allows affinity purification of recombinant protein with a poly-his-tag. Cell lysate containing the his-tagged recombinant protein among many other proteins is loaded to a Ni- or Co-affnity column and eluted with 50-3000 mM imidazole, depending on the binding strength of the his-tagged protein. It usually results in relatively pure protein when the recombinant protein is expressed in prokaryotic organisms. Purification of his-tagged protein from higher organisms such as yeasts or other eukaryotes may require a tandem affinity purification using two tags to yield higher purity. Alternatively, immobilized cobalt ions rather than nickel ions generally yields a substantial increase in purity and requires lower imidazole concentrations for elution of the his-tagged protein.

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