Avi-tagged Proteins Overview

Avi-tagged Proteins is a commonly used protein engineering technique for introducing biotin tags onto specific proteins. Usually located at the N-terminal or C-terminal of the protein, it has high specificity and affinity and can interact with biotin ligase (BirA enzyme).

AviTag tag protein is a short peptide consisting of 15 amino acids, the sequence is Gly-Leu-Asn-Asp-Ile-Phe-Glu-Ala-Gln-Lys-Ile-Glu-Trp-His-Glu, with a single biological Chlorinated lysine sites, completely different from known naturally biotinylatable sequences, can be added at the N- and C-termini of target proteins. After fusion expression, it can be biotinylated by biotin ligase. In order to purify recombinant protein, low-affinity monomeric avidin or avidin derivatives are used. In addition to protein separation and purification, it is also used for protein interaction effect research.

The Avi Tag labeling system has the following advantages:

1. Whether in vitro or in vivo, almost all proteins can be easily and efficiently biotinylated at a unique Avi Tag site
2. Biotinylation is achieved through the reaction of the enzyme and the substrate, the reaction conditions are quite mild and the specificity of the target label is extremely high
3. Biotin AviTag has only 15 amino acids, which has very little effect on the protein spatial structure

By introducing an Avi tag, biotin can be covalently attached to the Avi tag of a protein using a biotin ligation reaction catalyzed by the BirA enzyme. This labeling process is simple and flexible, and does not require complicated chemical modification and covalent binding steps.

The Versatile Applications of Avi-tag

The Avi-Tag system offers a straightforward and effective method for protein purification and immobilization on various surfaces. Due to its small size, the Avi-tag label can efficiently anchor proteins to different media, such as microtiter plates, chromatography media, biosensor chips, nitrocellulose membranes, and PVDF membranes. This versatile technology finds extensive use in medical and industrial sectors, with increasing applications in biosensing, diagnostics, scintillation proximity analysis, drug screening, and more. Avi-tag has demonstrated its exceptional utility in the following areas:

1. Protein purification: Avi-tag fusion protein purification using monomeric anti-biotin proves more efficient than other commonly used tagging methods, such as 6xHis.
2. Protein detection: After binding with avidin or streptavidin, Avi-tag fusion proteins can be detected using various assays, including Western blot and T cell staining.
3. Cell sorting: Avi-tag finds use in cell sorting applications.
4. Sensitive protein detection: Immobilized Avi-tag fusion proteins are widely employed, ranging from high-throughput screening to surface plasmon resonance detection of protein-protein interactions.

The biotin tagging of Avi-tagged proteins offers diverse applications and advantages. Firstly, biotin exhibits strong binding affinity with avidin, ensuring stable attachment of protein tags to other molecules or immobilized substrates. Secondly, Avi-tagged proteins can be combined with biotin-labeled ligands to investigate protein interactions, functional regulation, and signal transduction. This adaptable technology continues to pave the way for significant advancements in various research fields.

Research field of Avi-tagged Proteins

Avi-tagged Proteins are versatile and flexible and can be used in both in vitro and in vivo experiments. By introducing an Avi tag at a specific position of the protein, biotin labeling can be conveniently introduced to the protein of interest, allowing it to be located, tracked and studied.

The application of Avi-tag protein in the research field includes but not limited to the following aspects:

1. Protein expression and purification: Avi-tag protein can be combined with avidin in genetic engineering, and the high affinity and specificity of avidin can be used to facilitate efficient protein expression and purification. Through this labeling technology, the protein purification steps can be simplified, and the purity and yield can be improved.
2. Protein-protein interaction research: Avi-tag protein can be used as a tool for studying protein-protein interaction. The Avi-tag tag is introduced into the target protein, and then combined with avidin to bind with other proteins or ligands to study the interaction between proteins.
3. Research on cell signaling pathways: In the research of cell signaling pathways, Avi-tag proteins are widely used to study receptor-ligand interactions, signal transduction processes, and localization of intracellular proteins.
4. Drug screening: Avi-tag protein can be used as a target for drug screening and used in high-throughput screening to find potential drug candidates.
5. Immunological research: Avi-tag protein can also be used in immunological research, such as the study of antigen-antibody interaction in the immune response or the binding of T cell receptors to antigens.

In summary, Avi-tagged Proteins provide an easy and efficient method to introduce biotin tags into specific proteins. This technique has broad application prospects in biological research, protein engineering, and biomedical applications, providing a powerful tool for protein interaction, functional studies, and analysis.

References:

[1] Huang C, Jacobson K. Detection of protein-protein interactions using nonimmune IgG and BirA-mediated biotinylation. Biotechniques. 2010;49(6):881-886. doi:10.2144/000113550