The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Epitope tags are crucial for protein analysis but often have limited versatility.
• Existing tags like HA, FLAG®, and myc-tag present application-specific limitations.

Purpose of the Study:

• To introduce the ALFA-tag, a novel epitope tag designed for broad applicability in life sciences.
• To demonstrate the ALFA-tag's superior performance compared to established tags.

Main Methods:

• Characterization of the ALFA-tag's structure and function in prokaryotic and eukaryotic systems.
• Development and characterization of a high-affinity nanobody (NbALFA) against the ALFA-tag.
• Crystallographic analysis of the ALFA-tag and nanobody complex.
• Engineering of a nanobody mutant (NbALFA^PE) for peptide elution-based purification.

Main Results:

• The ALFA-tag forms a stable α-helix functional across diverse host systems and protein positions.
• NbALFA exhibits low picomolar affinity for ALFA-tagged proteins, suitable for microscopy, immunoprecipitation, and Western blotting.
• In vivo protein detection is achievable with the ALFA-tag system.
• NbALFA^PE enables efficient one-step purification of native proteins, complexes, and cells under physiological conditions.

Conclusions:

• The ALFA-tag offers enhanced versatility and performance over traditional epitope tags.
• The ALFA-tag system, coupled with NbALFA and NbALFA^PE, provides a powerful toolkit for protein detection, manipulation, and purification.