Novel His-tag Variants for Insertion Inside Polypeptide Chain.

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Hexahistidine (His-tag) affinity purification is a standard technique.
• N- or C-terminal His-tag fusion can negatively affect protein structure and function.
• Internal tag insertion is explored to mitigate N/C-terminal fusion issues.

Purpose of the Study:

• To design and characterize novel internal His-tags.
• To minimize steric hindrance and impact on protein structure/function.
• To broaden the utility of fusion proteins like eGFP and GrAD207.

Main Methods:

• Designed internal His-tags with two histidine triplets separated by glycine residues.
• Tested tag applicability by fusing them internally into enhanced green fluorescent protein (eGFP).
• Validated tag functionality with a modified GroEL chaperone domain (GrAD207).

Main Results:

• Successfully incorporated internal His-tags into eGFP and GrAD207.
• Demonstrated that internal tags minimize steric hindrance compared to terminal tags.
• Showed the potential for broad applicability across different protein classes.

Conclusions:

• Internal His-tags offer a viable alternative to N/C-terminal fusions.
• The designed tags can be adjusted for efficient purification of diverse proteins.
• This approach broadens the functionality of fusion proteins without compromising termini.