Chromatography of Proteins

Chromatography of Proteins

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Protein isolation using a His-Tag

A wide range of methods is available for isolating proteins, e.g. isolation by means of a genetic fusion of the desired protein with a hexapeptide of the amino acid histidine. This fusion enables the fusion protein to be purified via affinity chromatography.

In this process, the DNA sequence of the protein to be isolated is supplemented at the C or N terminus by the codons of a His tag (His linker, consisting of six histidine residues).

This His-Tag specifically binds to a column material with divalent nickel ions. DasNi2+-Ion is bound in an octahedral complex with the carrier material and water molecules. These can be displaced by histidine, creating a very stable chelate complex. Ni2+ is bound on these columns by nitrilotriacetic acid residues and can interact with two histidine residues of the protein in exchange for water. This specificity ensures that only the fusion protein binds to the column material.

The fusion protein is eluted with imidazole, which competitively displaces the histidine of the protein from the chelate complex. Alternatively, a low pH buffer can be used, which causes the protonation of the imidazole ring in the histidine. The nitrogen contained can then no longer function as a coordination partner.

For the expression of the protein to be isolated with a His-linker, special vector systems with strong promoters (partly available as kits) are available, into which the relevant gene can be cloned. The cloning must, however, be carried out with pinpoint accuracy so that the reading frames of the protein and the His-linker run into one another and so that the correct attachment of the histidine codons is guaranteed.

Precise cloning can be achieved with the aid of PCR, for example by adding the sequence of the His-tag to the nucleotide sequence of the protein to be isolated with the aid of modified primers.

Video: Αλλοίωση της δομής των πρωτεϊνών (July 2022).


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