Poly His tag protein expression system. Top question, Top Answer : Biotechnology course

Poly His tag protein expression system. Top question, Top Answer : Biotechnology course, Bio Data Technology: Bioinformatics and Biotechnology directory

poly histidine tag is one of highly usage technique in protein expression and protein purification techniques in biotechnology and genetic engineering laboratory. This also known as His-Tag, which is a string of usually between six and nine histidine residues in a chain. This method of tagging is especially useful as it allows for easy purification and detection of the recombinant protein.

How does his tag purification work?

His-tag purification uses the purification technique of immobilized metal affinity chromatography, or IMAC. In this technique, transition metal ions are immobilized on a resin matrix using a chelating agent such as iminodiacetic acid.

How many kDa is a His-tag?

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

What does a His-tag bind to?

Poly-His tags bind best to IMAC resins in near-neutral buffer conditions (physiologic pH and ionic strength). A typical binding/wash buffer consists of Tris-buffer saline (TBS) pH 7.2, containing 10-25 mM imidazole.

How do you add a His-tag?

(A) The His-tag is added by inserting the DNA encoding a protein of interest in a vector that has the tag ready to fuse at the C-terminus. (B) The His-tag is added using primers containing the tag, after a PCR reaction the tag gets fused to the N-terminus of the gene.

How do you purify his tag protein?

His-tagged proteins can be purified by a single-step affinity chromatography, namely immobilized metal ion affinity chromatography (IMAC), which is commercially available in different kinds of formats, Ni-NTA matrices being the most widely used.

How does the His tag bind to the nickel column?

The His tag binds to divalent cations immobilized on metal chelation resin, such as nickel resin Ni-NTA (Qiagen GmbH, Germany) or cobalt resin TALON (Clontech, GmbH, Germany). Under our purification conditions (see below) cobalt beads give better results than nickel beads.

What is the usual way of eluting His-tagged proteins?

imidazole

Elution and recovery of captured His-tagged protein from an IMAC column is accomplished by using a high concentration of imidazole (at least 200 mM), low pH (e.g., 0.1 M glycine-HCl, pH 2.5) or an excess of strong chelators (e.g., EDTA). Imidazole is the most common elution agent.

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