Difference between revisions of "Part:BBa K2255003:Design"

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===Design Notes===
 
===Design Notes===
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We wanted to create a multi-tag with a his-tag (attachment to Co or Ni column) and a streptavidine-tag (wich can bind biotin). Between those tag we add a TEV clivation site in order to give the possibility of decrease the lengh of this multi-tag.  
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We wanted to create a multi-tag with a his-tag (attachment to Co or Ni column) and a Strep-tag <ref name= Strep>Schmidt, Thomas GM; Skerra, Arne (2007). "The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins". Nature Protocols. 2 (6): 1528–35. PMID 17571060. doi:10.1038/nprot.2007.209</ref> (wich has an affinity towards Strep-Tactin®). Between those tag, we added a TEV <ref name=TEV>Parks TD, Leuther KK, Howard ED, Johnston SA, Dougherty WG (February 1994). "Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase". Anal. Biochem. 216 (2): 413–7. PMID 8179197. doi:10.1006/abio.1994.1060</ref> cutting site to give the possibility to decrease the length of this multi-tag.  
  
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Our tag is composed of : StrepTag-TEV-6His.
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Our tag is composed of StrepTag-TEV-6His, which can be constructed as GSG-WSHPQPEL-GSG-ASQFYLNE-GSG-HHHHHH
  
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Which can be constructed as : GSG-WSHPQPEL-GSG-ASQFYLNE-GSG-HHHHHH
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Glycine and serine residues are added to the sequence in order to give flexibility and space between the tags and the cutting site.
  
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Glycin and serine residues are added to the sequence in order to give flexibility and space between tags and clivation site.
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We retro-translated this sequence and optimized it for ''E. coli''.
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We retro-translate this sequence and optimized it for ''E. coli''.
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===Source===
 
===Source===
  
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This part is made with the consensus sequence of streptavidine tag , histidine tag and TEV protease clivation site.
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This part is made with the consensus sequence of the streptavidine tag, the histidine tag and the TEV protease cutting site.
  
 
===References===
 
===References===
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<references/>

Latest revision as of 11:31, 6 October 2017


Multi-Tag (Rfc 25)


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]


Design Notes

We wanted to create a multi-tag with a his-tag (attachment to Co or Ni column) and a Strep-tag [1] (wich has an affinity towards Strep-Tactin®). Between those tag, we added a TEV [2] cutting site to give the possibility to decrease the length of this multi-tag.

Our tag is composed of StrepTag-TEV-6His, which can be constructed as GSG-WSHPQPEL-GSG-ASQFYLNE-GSG-HHHHHH

Glycine and serine residues are added to the sequence in order to give flexibility and space between the tags and the cutting site.

We retro-translated this sequence and optimized it for E. coli.

Source

This part is made with the consensus sequence of the streptavidine tag, the histidine tag and the TEV protease cutting site.

References

  1. ↑ Schmidt, Thomas GM; Skerra, Arne (2007). "The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins". Nature Protocols. 2 (6): 1528–35. PMID 17571060. doi:10.1038/nprot.2007.209
  2. ↑ Parks TD, Leuther KK, Howard ED, Johnston SA, Dougherty WG (February 1994). "Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase". Anal. Biochem. 216 (2): 413–7. PMID 8179197. doi:10.1006/abio.1994.1060