Part:BBa_K5160011

3xHA

Overview

The HA tag (HA-tag) is a protein tag based on the human influenza hemagglutinin (HA) antigen, with its chemical essence being a short amino acid sequence derived from the 98-106th amino acids of the human influenza HA molecule, specifically YPYDVPDYA. The HA tag is widely used as an epitope tag in expression vectors, facilitating protein detection, separation, and purification, as it does not interfere with the protein's biological activity or distribution. By adding the HA tag to the target protein, specific antibodies against the HA tag can bind to the recombinant protein, enabling convenient experiments such as immunohistochemistry (IHC) and Western Blotting. HA antibodies can also be immobilized on agarose beads for protein purification, enhancing purification efficiency. Many recombinant proteins can express the HA tag, aiding in the rapid acquisition of information regarding their localization, expression, or biological functions.
In project design, to purify and identify the target protein while enhancing the detection signal and improving purification efficiency, we attach three HA tag proteins to the C-terminus of the target protein to create the corresponding vector. Subsequently, we introduce the target gene into the tomato genome sequence through transient infection technology or transgenic technology. When the target gene is expressed, the HA tag protein is also expressed, which can be used for subsequent Western blot (WB) detection and co-immunoprecipitation to verify the expression of the target protein.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 58
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design

1. During the design process, considering the need to add the vacuolar signal peptide SPS-NTPP to the N-terminus of the target sweet protein, we deliberately placed the 3xHA tag at the C-terminus of the target protein to prevent it from interfering with the SPS-NTPP vacuolar signal peptide.
2. Meanwhile, to improve the accuracy of detection, we used three HA tags. Multiple tags can increase the number of antigenic epitopes, thereby enhancing the chances of recognition by antibodies and strengthening the signal intensity of Western blot (WB) or immunoprecipitation. Additionally, multiple tags can reduce the occurrence of false-negative results in experiments. Even if one tag fails to be detected due to certain reasons, other tags may still be detectable. Furthermore, multiple tags can enhance the detection signal, improve purification efficiency, increase flexibility, reduce the risk of loss, and facilitate quantitative analysis.

Source

The HA tag originates from the 98-106th amino acid residues of the human influenza virus HA molecule and is a polypeptide with a length of 9 amino acids. The molecular weight of the HA tag is relatively small, exerting minimal influence on the spatial structure of the target protein and not interfering with the protein's biological activity or biodistribution.