Difference between revisions of "Part:BBa K1150016"
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1150016 short</partinfo>
 
<partinfo>BBa_K1150016 short</partinfo>
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{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
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! colspan="2" style="background:#FFBF00;"|HA tag
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|-
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|'''Function'''
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|Tag
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|-
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|'''Use in'''
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|Mammalian cells
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|-
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|'''RFC standard'''
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|[https://parts.igem.org/Help:Assembly_standard_25 RFC 25]
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|-
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|'''Backbone'''
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|pSB1C3<br>
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|-
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|'''Submitted by'''
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|[http://2013.igem.org/Team:Freiburg Freiburg 2013]
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|}
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The human influenza hemagglutinin (HA)–tag enables the detection, isolation and purification of proteins. <br> Engineered into recombinant proteins, it does not seem to interfere with its bioactivity or biodistribution. <br> It is derived from a surface glycoprotein of the human virus. The tag consists only of the amino acids 98-106 of the original protein. <br><br>
 
The human influenza hemagglutinin (HA)–tag enables the detection, isolation and purification of proteins. <br> Engineered into recombinant proteins, it does not seem to interfere with its bioactivity or biodistribution. <br> It is derived from a surface glycoprotein of the human virus. The tag consists only of the amino acids 98-106 of the original protein. <br><br>
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===Usage and Biology===
 
===Usage and Biology===
−
This biobrick constitutes the improvement of BBa_K128001 containing the HA-tag in the silver standard. As the RFC23 pre- and suffix do not automatically include a Start and Stop codon respectively ,N- and C-terminal fusions of this HA-tag lead not automatically to a transcriptional Start and Stop of the corresponding DNA-sequence. In contrast, the RFC25 biobrick (which is also design for functional part fusions) has the start codon and stop codon automatically included in its prefix and suffix.  
+
This BioBrick constitutes the improvement of BBa_K128001 containing the HA-tag in silver standard (for further information click [https://parts.igem.org/Part:BBa_K128001:Experience] or [http://2013.igem.org/Team:Freiburg/parts/improvement]). As the RFC23 pre- and suffix do not automatically include a start and stop codon respectively, N- and C-terminal fusions of this HA-tag don't lead automatically to a transcriptional start and stop of the corresponding DNA-sequence. In contrast, the RFC25 biobrick (which is also suitable for functional part fusions) has the start codon and stop codon automatically included in its prefix and suffix.  
  
−
As our improved BBa_K1150016 contains the RFC25 prefix, start or stop codon are given beforehand which spends  time and facilitats cloning.   
+
As our improved BBa_K1150016 contains the RFC25 prefix and suffix, start or stop codon are given beforehand which facilitates cloning and saves time.   
  
−
The new version includes the same sequence of amino and has the following advantages:
+
The new version includes the same sequence of amino acids and has the following advantages:
  
 
• Cloning the HA-tag BBa_K1150016 N-terminal to other fusion parts leads to an automatic transcriptional start due to the introduced ATG in the RFC25 prefix.
 
• Cloning the HA-tag BBa_K1150016 N-terminal to other fusion parts leads to an automatic transcriptional start due to the introduced ATG in the RFC25 prefix.
−
• Cloning the HA-tag BBa_K1150016 C-terminal to other fusion parts leads to an automatic transcriptional stop due to the introduced Stopp-Codon in the RFC25 suffix.
 
  
−
[[File:HA_neu_2_Freigem_2013.png|300px|]]
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• Cloning the HA-tag BBa_K1150016 C-terminal to other fusion parts leads to an automatic transcriptional stop due to the introduced stop codon in the RFC25 suffix.
  
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'''Fig.2:''' BBa_K1150016 with RFC25 prefix and suffix.
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[[File:HA_neu_2_Freigem_2013.png|900px|]]
  
−
Before bringing this tag into the iGEM-standard we first tested its functionality via Western blot detection, see Figure 2. As it worked fine in mammalian cells, we used this sequence for our standardized construct. Testing of our standardized devices including the BBa_K1150016 worked well and also Western blot detection was fine, see Fig.3.
+
'''Figure 2:''' BBa_K1150016 with RFC25 prefix and suffix.
  
−
[[File:HA_neu_nicht_standardisiert_Freigem_2013.png|100px|]]
+
Before bringing this tag into the iGEM-standard we first tested its functionality via western blot detection, see Figure 2. As it worked fine in mammalian cells, we used this sequence for our standardized constructs. Testing of our standardized devices including the BBa_K1150016 worked well and also western blot detection was good, see Fig.3.
 +
 
 +
[[File:HA_neu_nicht_standardisiert_Freigem_2013.png|200px|]]
  
 
'''Figure 3:''' HA-tag fused to CRY2-C-RAF-CIBN-protein encoded on the pEF6/V5-His-TOPO plasmid.
 
'''Figure 3:''' HA-tag fused to CRY2-C-RAF-CIBN-protein encoded on the pEF6/V5-His-TOPO plasmid.
  
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[[File:HA_tag_neu_standaridisiert_%28CMV-Cas-VP16%29_Freigem2013.png|100px|]]
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[[File:UniBAssblotfertig2.1-team_Freiburgforregistry.PNG|800px|]]
  
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'''Figure 4.:''' HA-tag fused to the Cas-VP16-protein encoded on the RFC25 pSB1C3 backbone.
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'''Figure 4:''' HA-tag fused to different dCas9-fusion proteins encoded on the RFC25 pSB1C3 backbone.
  
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<span class='h3bb'>Sequence and Features</span>
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===Sequence and Features===
 
<partinfo>BBa_K1150016 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1150016 SequenceAndFeatures</partinfo>
  
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<partinfo>BBa_K1150016 parameters</partinfo>
 
<partinfo>BBa_K1150016 parameters</partinfo>
 
<!-- -->
 
<!-- -->
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=Contribution : iGEM22_CSMU_Taiwan=
 +
<li>
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<b>Group:</b> iGEM22_CSMU_Taiwan
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</li>
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<li>
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<b>Author:</b> LIN, ZONG-HAN, HSIAO, WEI-CHENG
 +
</li>
 +
<li>
 +
<b>Summary:</b>
 +
</li>
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HA-tag is a fragment of peptide originated from Human influenza hemagglutinin. In our construct design, we place HA-tag after our target protein, AID, so as to conduct following experiments such as Western-blot, which confirm that our target protein is expressed. It is indeed a useful tool to verify the target protein!
 +
 +
===References===
 +
Saiz-Baggetto S, Méndez E, Quilis I, Igual JC, Bañó MC. Chimeric proteins tagged with specific 3xHA cassettes may present instability and functional problems. PLoS One. 2017 Aug 11;12(8):e0183067. doi: 10.1371/journal.pone.0183067. PMID: 28800621; PMCID: PMC5553802.

Latest revision as of 06:47, 10 October 2022

HA tag

HA tag
Function Tag
Use in Mammalian cells
RFC standard RFC 25
Backbone pSB1C3
Submitted by [http://2013.igem.org/Team:Freiburg Freiburg 2013]


The human influenza hemagglutinin (HA)–tag enables the detection, isolation and purification of proteins.
Engineered into recombinant proteins, it does not seem to interfere with its bioactivity or biodistribution.
It is derived from a surface glycoprotein of the human virus. The tag consists only of the amino acids 98-106 of the original protein.

The HA-tag was used mainly for protein detection. After blotting our SDS-PAGE gels on PVDF membranes, anti-HA-antibodies were used to target our recombinant proteins. Secondary antibodies furnished proof of our proteins.

Ha-tag_registry_freiburg_13.PNG
Figure 1: Proteins with HA-tag were detected with anti-HA-antibody on a Western Blot.

Usage and Biology

This BioBrick constitutes the improvement of BBa_K128001 containing the HA-tag in silver standard (for further information click [1] or [http://2013.igem.org/Team:Freiburg/parts/improvement]). As the RFC23 pre- and suffix do not automatically include a start and stop codon respectively, N- and C-terminal fusions of this HA-tag don't lead automatically to a transcriptional start and stop of the corresponding DNA-sequence. In contrast, the RFC25 biobrick (which is also suitable for functional part fusions) has the start codon and stop codon automatically included in its prefix and suffix.

As our improved BBa_K1150016 contains the RFC25 prefix and suffix, start or stop codon are given beforehand which facilitates cloning and saves time.

The new version includes the same sequence of amino acids and has the following advantages:

• Cloning the HA-tag BBa_K1150016 N-terminal to other fusion parts leads to an automatic transcriptional start due to the introduced ATG in the RFC25 prefix.

• Cloning the HA-tag BBa_K1150016 C-terminal to other fusion parts leads to an automatic transcriptional stop due to the introduced stop codon in the RFC25 suffix.

HA neu 2 Freigem 2013.png

Figure 2: BBa_K1150016 with RFC25 prefix and suffix.

Before bringing this tag into the iGEM-standard we first tested its functionality via western blot detection, see Figure 2. As it worked fine in mammalian cells, we used this sequence for our standardized constructs. Testing of our standardized devices including the BBa_K1150016 worked well and also western blot detection was good, see Fig.3.

HA neu nicht standardisiert Freigem 2013.png

Figure 3: HA-tag fused to CRY2-C-RAF-CIBN-protein encoded on the pEF6/V5-His-TOPO plasmid.

UniBAssblotfertig2.1-team Freiburgforregistry.PNG

Figure 4: HA-tag fused to different dCas9-fusion proteins encoded on the RFC25 pSB1C3 backbone.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Contribution : iGEM22_CSMU_Taiwan

  • Group: iGEM22_CSMU_Taiwan
  • Author: LIN, ZONG-HAN, HSIAO, WEI-CHENG
  • Summary:

    • HA-tag is a fragment of peptide originated from Human influenza hemagglutinin. In our construct design, we place HA-tag after our target protein, AID, so as to conduct following experiments such as Western-blot, which confirm that our target protein is expressed. It is indeed a useful tool to verify the target protein!

    References

    Saiz-Baggetto S, Méndez E, Quilis I, Igual JC, Bañó MC. Chimeric proteins tagged with specific 3xHA cassettes may present instability and functional problems. PLoS One. 2017 Aug 11;12(8):e0183067. doi: 10.1371/journal.pone.0183067. PMID: 28800621; PMCID: PMC5553802.