HA-tag

Last updated
12CA5 Fv-clasp fragment (green, cyan) with its antigen peptide, YPYDVPDYA (magenta), which is used as HA tag. Image created with PyMOL from PDB: 5XCS . HA tag.png
12CA5 Fv-clasp fragment (green, cyan) with its antigen peptide, YPYDVPDYA (magenta), which is used as HA tag. Image created with PyMOL from PDB: 5XCS .

The HA-tag is a protein tag derived from the human influenza hemagglutinin (HA) protein, which allows the virus to target and enter host cells. An HA-tag is composed of a peptide derived from the HA-molecule corresponding to amino acids 98-106, which can be recognized and selectively bound by commercially available antibodies. This makes HA a powerful tool in molecular biology, commonly included in expression vectors and in the production of recombinant proteins. [1] Like other epitope tags, HA-tag is small and generally does not alter the traits of proteins it is attached to. [2] As a result HA-tags are often used to identify protein-protein interactions or to detect protein expression, using Co-Immunoprecipitation or Western blot respectively. [3] [4]

Contents

The HA-tag is not suitable for detection or purification of proteins from apoptotic cells since it is cleaved by Caspase-3 and / or Caspase-7 after its sequence DVPD, causing it to lose its immunoreactivity. [5] Labeling of endogenous proteins with HA-tag using CRISPR was recently accomplished in-vivo in differentiated neurons. [6]

Sequence

The DNA sequences for the HA-tag include: 5'-TAC-CCA-TAC-GAT-GTT-CCA-GAT-TAC-GCT-3' or 5'-TAT-CCA-TAT-GAT-GTT-CCA-GAT-TAT-GCT-3'. The resulting amino acid sequence is YPYDVPDYA (Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala). [7]

See also

Related Research Articles

<span class="mw-page-title-main">Adenylyl cyclase</span> Enzyme with key regulatory roles in most cells

Adenylate cyclase is an enzyme with systematic name ATP diphosphate-lyase . It catalyzes the following reaction:

An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that binds to the epitope is called a paratope. Although epitopes are usually non-self proteins, sequences derived from the host that can be recognized are also epitopes.

<span class="mw-page-title-main">Protein splicing</span> The post-translational removal of peptide sequences from within a protein sequence

Protein splicing is an intramolecular reaction of a particular protein in which an internal protein segment is removed from a precursor protein with a ligation of C-terminal and N-terminal external proteins on both sides. The splicing junction of the precursor protein is mainly a cysteine or a serine, which are amino acids containing a nucleophilic side chain. The protein splicing reactions which are known now do not require exogenous cofactors or energy sources such as adenosine triphosphate (ATP) or guanosine triphosphate (GTP). Normally, splicing is associated only with pre-mRNA splicing. This precursor protein contains three segments—an N-extein followed by the intein followed by a C-extein. After splicing has taken place, the resulting protein contains the N-extein linked to the C-extein; this splicing product is also termed an extein.

<span class="mw-page-title-main">Alfred G. Gilman</span> American pharmacologist

Alfred Goodman Gilman was an American pharmacologist and biochemist. He and Martin Rodbell shared the 1994 Nobel Prize in Physiology or Medicine "for their discovery of G-proteins and the role of these proteins in signal transduction in cells."

Protein tags are peptide sequences genetically grafted onto a recombinant protein. Tags are attached to proteins for various purposes. They can be added to either end of the target protein, so they are either C-terminus or N-terminus specific or are both C-terminus and N-terminus specific. Some tags are also inserted at sites within the protein of interest; they are known as internal tags.

FLAG-tag, or FLAG octapeptide, or FLAG epitope, is a peptide protein tag that can be added to a protein using recombinant DNA technology, having the sequence DYKDDDDK. It is one of the most specific tags and it is an artificial antigen to which specific, high affinity monoclonal antibodies have been developed and hence can be used for protein purification by affinity chromatography and also can be used for locating proteins within living cells. FLAG-tag has been used to separate recombinant, overexpressed protein from wild-type protein expressed by the host organism. FLAG-tag can also be used in the isolation of protein complexes with multiple subunits, because FLAG-tag's mild purification procedure tends not to disrupt such complexes. FLAG-tag-based purification has been used to obtain proteins of sufficient purity and quality to carry out 3D structure determination by x-ray crystallography.

Michael Howard Wigler is an American molecular biologist who has directed a laboratory at Cold Spring Harbor Laboratory since 1978 and is a member of the National Academy of Sciences. He is best known for developing methods to genetically engineer animal cells and his contributions to cancer, genomics and autism genetics.

<span class="mw-page-title-main">ADCY6</span> Protein-coding gene in humans

Adenylyl cyclase type 6 is an enzyme that in humans is encoded by the ADCY6 gene.

<span class="mw-page-title-main">ADCY3</span> Protein-coding gene in the species Homo sapiens

Adenylyl cyclase type 3 is an enzyme that in humans is encoded by the ADCY3 gene.

<span class="mw-page-title-main">ADCY5</span> Protein-coding gene in the species Homo sapiens

Adenylyl cyclase type 5 is an enzyme that in humans is encoded by the ADCY5 gene.

<span class="mw-page-title-main">ADCY1</span> Protein-coding gene in the species Homo sapiens

Adenylyl cyclase type 1 is an enzyme that in humans is encoded by the ADCY1 gene.

<span class="mw-page-title-main">CAP1</span> Gene of the species Homo sapiens

Adenylyl cyclase-associated protein 1 is an enzyme that in humans is encoded by the CAP1 gene.

<span class="mw-page-title-main">ADCY2</span> Protein-coding gene in the species Homo sapiens

Adenylyl cyclase type 2 is an enzyme typically expressed in the brain of humans, that is encoded by the ADCY2 gene. It belongs to the adenylyl cyclase class-3 or guanylyl cyclase family because it contains two guanylate cyclase domains. ADCY2 is one of ten different mammalian isoforms of adenylyl cyclases. ADCY2 can be found on chromosome 5 and the "MIR2113-POU3F2" region of chromosome 6, with a length of 1091 amino-acids. An essential cofactor for ADCY2 is magnesium; two ions bind per subunit.

<span class="mw-page-title-main">ADCY7</span> Protein-coding gene in the species Homo sapiens

Adenylyl cyclase type 7 is an enzyme that in humans is encoded by the ADCY7 gene.

<span class="mw-page-title-main">ADCY9</span> Protein-coding gene in the species Homo sapiens

Adenylyl cyclase type 9 is an enzyme that in humans is encoded by the ADCY9 gene.

<span class="mw-page-title-main">ADCY8</span> Protein-coding gene in the species Homo sapiens

Adenylyl cyclase type 8 is an enzyme that in humans is encoded by the ADCY8 gene.

<span class="mw-page-title-main">ADCY4</span> Protein-coding gene in the species Homo sapiens

Adenylyl cyclase type 4 is an enzyme that in humans is encoded by the ADCY4 gene.

A killer yeast is a yeast, such as Saccharomyces cerevisiae, which is able to secrete one of a number of toxic proteins which are lethal to susceptible cells. These "killer toxins" are polypeptides that kill sensitive cells of the same or related species, often functioning by creating pores in target cell membranes. These yeast cells are immune to the toxic effects of the protein due to an intrinsic immunity. Killer yeast strains can be a problem in commercial processing because they can kill desirable strains. The killer yeast system was first described in 1963. Study of killer toxins helped to better understand the secretion pathway of yeast, which is similar to those of more complex eukaryotes. It also can be used in treatment of some diseases, mainly those caused by fungi.

Ras2 is a Saccharomyces cerevisiae guanine nucleotide-binding protein which becomes activated by binding GTP when glucose is present in the environment. It affects growth regulation and starvation response.

The NE-tag is a synthetic peptide tag designed as an epitope tag for detection, quantification and purification of recombinant protein. This patented peptide sequence is composed of eighteen hydrophilic amino acids. This short peptide does not adopt any significant homology to any existing proteins found in nature. This synthetic NE peptide adopts random coil conformation and showing strong immunogenicity. This is advantageous to offer stringent specificity to the NE-tagged proteins, which are readily to be detected, quantitated, and purified.

References

  1. Field J, Nikawa J, Broek D, MacDonald B, Rodgers L, Wilson IA, Lerner RA, Wigler M (1988). "Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method". Mol Cell Biol. 8 (5): 2159–65. doi:10.1128/mcb.8.5.2159-2165.1988. PMC   363397 . PMID   2455217.
  2. Kimple, Michelle E.; Brill, Allison L.; Pasker, Renee L. (2013-09-24). "Overview of Affinity Tags for Protein Purification". Current Protocols in Protein Science. 73: Unit–9.9. doi:10.1002/0471140864.ps0909s73. ISSN   1934-3655. PMC   4527311 . PMID   24510596.
  3. "Anti-HA Tag Antibody Updated 28/10/2021". Merck Millipore.
  4. "Overview of Epitope Tagging - US". www.thermofisher.com. Retrieved 2024-07-25.
  5. Schembri L, Dalibart R, Tomasello F, Legembre P, Ichas F, De Giorgi F (February 2007). "The HA tag is cleaved and loses immunoreactivity during apoptosis". Nature Methods. 4 (2): 107–8. doi:10.1038/nmeth0207-107. PMID   17264856. S2CID   32173039.
  6. Mikuni T, Nishiyama J, Sun Y, Kamasawa N, Yasuda R (2016). "High Throughput, High Resolution Mapping of Protein Localization in Mammalian Brain by In Vivo Genome Editing". Cell. 165 (7): 1803–1817. doi:10.1016/j.cell.2016.04.044. PMC   4912470 . PMID   27180908.
  7. "Addgene: HA-tag". www.addgene.org. Retrieved 2024-07-25.

Further reading