Does adding the His6 tag alter its mass substantially?
Adding a His6 tag to a protein is a common strategy in molecular biology to facilitate its purification and detection. The His6 tag is a short sequence of six histidine residues, which can be fused to the N- or C-terminus of a protein. This tag is highly attractive due to its strong affinity for immobilized metal ions, making it an ideal tool for protein purification using affinity chromatography. However, one of the most frequently asked questions in this field is whether adding the His6 tag substantially alters the mass of the protein. In this article, we will explore this issue and provide insights into the potential impact of the His6 tag on protein mass.
The His6 tag itself has a molecular weight of approximately 842.55 Daltons. Therefore, when fused to a protein, the total mass of the protein with the His6 tag is expected to increase by this amount. However, the actual impact on the protein’s mass can vary depending on several factors.
Firstly, the position of the His6 tag within the protein sequence plays a crucial role in determining the extent of mass alteration. When fused to the N-terminus, the His6 tag will add its mass directly to the protein, resulting in a more substantial increase in overall mass. Conversely, when fused to the C-terminus, the protein’s mass will increase by the same amount, but the impact on the protein’s overall structure and function may be less pronounced.
Secondly, the length of the protein itself can influence the mass alteration. Smaller proteins may experience a more significant percentage increase in mass when the His6 tag is added, while larger proteins may experience a smaller percentage increase. This is because the relative contribution of the His6 tag to the total protein mass is higher in smaller proteins.
Additionally, the presence of other tags or modifications in the protein can also affect the overall mass. For example, if the protein already contains a tag or a modification with a significant mass, the addition of the His6 tag may result in a relatively smaller increase in mass.
It is important to note that, despite the potential increase in mass, the His6 tag itself does not typically cause any significant structural or functional alterations to the protein. The histidine residues in the tag are generally well-folded and do not disrupt the protein’s native structure. This allows the protein to retain its biological activity and function even after the addition of the His6 tag.
In conclusion, while adding the His6 tag to a protein does result in a slight increase in mass, the impact is generally not substantial. The actual mass alteration depends on factors such as the position of the tag, the length of the protein, and the presence of other tags or modifications. Nonetheless, the His6 tag remains a valuable tool in protein purification and research, offering a reliable and efficient means to isolate and study proteins of interest.
