ProteinTags are classified as peptide sequence that have been incorporated into thegenome of a large variety of protein. In molecular biology, two of the mostcommonly used protein tags are Histidine tag and Green fluorescent protein (GFP)tags which are useful laboratory tools for the detection and purification ofrecombinant proteins. These tags have allowed researchers to further investigateessential characteristics of proteins such as localization, gene expression inliving and fixed organisms and make findings that have influence the sciencecommunity. A Histag is classified as a cleavable DNA sequence containing two to 10 histidineresidues, most commonly 6 residues, that is incorporated into a plasmid. His Tagsare small making them able to be incorporated into expression vectors and donot cause major adverse effects nor affect the properties of the recombinantprotein of interest if use properly.
His tags Beforepurification of a protein, the his tag are commonly incorporated into arecombinant proteins at the N-terminus or C-terminus of the protein ofinterest. 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 tothe transition metals for purification purpose if the folding of a protein hasocculded it. If this occurs moving the tag or conducting the purification undermore basic conditions will perturb contamination of the protein sample.
Histags are usually cleaved from the protein using site specific proteases, suchas thrombin and factor Xa.Histidineis basic amino acid that contains a positively charged imidazole functionalgroup, it exhibits a strong interaction with transition metals ions. Because ofthis property, Histidine tags are used with immobilized transition metalchromatography (IMAC) to separate the protein of interest for analyzationpurposes. The effectiveness of IMAC is dependent on the interaction of theproteins with transition metal ions that are present in the column resin.
Histidine’simidazole group acts as an electron donor and forms coordination bonds with theimmobilized metals which is present in the resin/column in the IMAC. The columnfor this type of chromatography is composed of transition metal ions commonlyNi2+ and Co2+. Histidine tags will bind to the resin as they pass and will beretained in the column. An elution buffer commonly containing free imidazolewill be applied to wash the resin and elute the retained histidine containingproteins are eluted.
This occurs because the free imidazole acts as a cationexchange. The number of histidine residues in the tag are variable. Even thoughthe protein containing his tag will have a high affinity for transition metalsas the number of residues increase or if two hexahistidine tags are used, it isadvised to use the shortest his tag in order to minimize the possible perturbation of protein function. In the majority of case, a 6 residuehis tag will suffice.