The His-tag, or Histidine-tag, is a short sequence of histidine amino acids used in molecular biology to simplify protein purification. Although a small addition to a protein, its specific molecular weight is a significant detail for scientists working with these molecules.
Understanding the His-Tag
A His-tag is composed of six to ten consecutive histidine residues. This tag is used because histidine exhibits a strong affinity for certain metal ions like nickel or cobalt. This characteristic is exploited in Immobilized Metal Affinity Chromatography (IMAC). In IMAC, proteins with a His-tag bind to a column containing immobilized metal ions, allowing other unbound cellular components to be washed away.
The genetic code for the His-tag is engineered directly into the DNA sequence of the gene encoding the protein of interest. This genetic fusion ensures that when the protein is produced within a cell, the His-tag is attached to either the N-terminus (beginning) or C-terminus (end) of the protein. This allows for straightforward purification of the tagged protein from complex biological mixtures. The presence of the tag does not interfere with the protein’s natural function.
Calculating His-Tag Molecular Weight
The molecular weight of a His-tag is determined by summing the molecular weights of its individual constituent amino acids. A single histidine residue has a molecular weight of 137.14 grams per mole (g/mol). This value accounts for the histidine side chain and the atoms from the polypeptide backbone that become part of the residue.
For a 6xHis-tag, the molecular weight is calculated by multiplying the molecular weight of one histidine residue by six. This results in a molecular weight of 822.84 g/mol for a 6xHis-tag. If a linker region is present between the His-tag and the protein of interest, its molecular weight also needs to be added to the total. This calculation is important for accurate protein characterization.
Why His-Tag Molecular Weight Matters in Research
Knowing the molecular weight of a His-tag is important because it directly contributes to the overall molecular weight of the tagged protein. This contribution is relevant in various biochemical techniques used to analyze proteins. For example, in Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), which separates proteins by size, the His-tag adds a small but discernible increase to the protein’s apparent molecular weight. Ignoring this addition would lead to an underestimation of the true molecular weight of the protein itself.
Similarly, in mass spectrometry, a technique used for accurate mass determination of molecules, the His-tag’s molecular weight must be accounted for to identify and characterize the protein of interest. If the His-tag’s mass is not factored into the calculations, researchers could misinterpret their data, potentially leading to incorrect conclusions about the protein’s identity, modifications, or purity. Therefore, incorporating the His-tag’s molecular weight into experimental analysis is a standard practice for maintaining accuracy in protein research.