Protein
Tags are classified as peptide sequence that have been incorporated into the
genome of a large variety of protein. In molecular biology, two of the most
commonly used protein tags are Histidine tag and Green fluorescent protein (GFP)
tags which are useful laboratory tools for the detection and purification of
recombinant proteins. These tags have allowed researchers to further investigate
essential characteristics of proteins such as localization, gene expression in
living and fixed organisms and make findings that have influence the science
community.

 

A His
tag is classified as a cleavable DNA sequence containing two to 10 histidine
residues, most commonly 6 residues, that is incorporated into a plasmid. His Tags
are small making them able to be incorporated into expression vectors and do
not cause major adverse effects nor affect the properties of the recombinant
protein of interest if use properly. His tags

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Before
purification of a protein, the his tag are commonly incorporated into a
recombinant proteins at the N-terminus or C-terminus of the protein of
interest. The optimal placement of the tag, either N or C, is protein specific.

However if the tag is placed inproperly, the tag may not be accessible to the to
the transition metals for purification purpose if the folding of a protein has
occulded it. If this occurs moving the tag or conducting the purification under
more basic conditions will perturb contamination of the protein sample. His
tags are usually cleaved from the protein using site specific proteases, such
as thrombin and factor Xa.

Histidine
is basic amino acid that contains a positively charged imidazole functional
group, it exhibits a strong interaction with transition metals ions. Because of
this property, Histidine tags are used with immobilized transition metal
chromatography (IMAC) to separate the protein of interest for analyzation
purposes. The effectiveness of IMAC is dependent on the interaction of the
proteins with transition metal ions that are present in the column resin. Histidine’s
imidazole group acts as an electron donor and forms coordination bonds with the
immobilized metals which is present in the resin/column in the IMAC. The column
for this type of chromatography is composed of transition metal ions commonly
Ni2+ and Co2+. Histidine tags will bind to the resin as they pass and will be
retained in the column. An elution buffer commonly containing free imidazole
will be applied to wash the resin and elute the retained histidine containing
proteins are eluted. This occurs because the free imidazole acts as a cation
exchange. The number of histidine residues in the tag are variable. Even though
the protein containing his tag will have a high affinity for transition metals
as the number of residues increase or if two hexahistidine tags are used, it is
advised to use the shortest his tag in order to minimize the possible perturbation of protein function. In the majority of case, a 6 residue
his tag will suffice.

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