Design a DNA sequence to introduce 4 copies of tandem HA tag

Design a DNA sequence to introduce 4 copies of tandem HA tags on N-terminus of the following hypothetical protein. How will you apply this tag to detect your protein in a mix?

Solution

Answer:

The HA tag is derived from the HA-molecule (Human influenza agglutinin) corresponding to amino acids 98-106. It has been extensively used as a general epitope tag in expression vectors.

5\' ---(RE1)- (TAC CCA TAC GAT GTT CCA GAT TAC GCT)₄ - (RE2) - Start - Protein of interest - Stop - (RE3)--- 3\'

1. The dotted lines at the 5\' and 3\' end designates the vector sequences.

2. RE1, RE2 and RE3 are the restriction sites.

3. The 4 tandem HA tag sequences have been placed in braces with 4 at the subscript.

4. Linkers can be added in between the tandem HA tags if desired.

Purification:

The tagged protein can be purified from a mixture of proteins by passing the supernatant through an anti-HA agarose column.  The anti-HA antibody coupled to the resin is a high-affinity mouse IgG1 monoclonal antibody that recognizes the HA-epitope tag.

The protein may be eluted by adding one bed volume of non-reducing 2X SDS-PAGE loading buffer.  If the eluted HA-tagged protein is to be used for function applications, then the protein can be eluted with HA Peptide. It can also be eluted by cleaving with Caspase-3 and / or Caspase-7 after its sequence DVPD, resulting in its loss of immunogenecity.