Part:BBa_K2586004

Strep-Tag-aroE: aroE linked to a Strep-Tag

This part is the combination of (BBa_K2586003) with a Strep-Tag.
The Strep-Protein is useful as it allows the purification of the tagged protein, to carry out further interaction studies. The Strep-protein can also be used in (SPINE): Strep-protein interaction experiments. SPINE is a powerful tool to detect protein-protein interactions in vivo (Herzberg et. al. 2007). The bait protein (aroE in this case) has to be expressed with a Strep-tag under the conditions when the potential interaction partners are presumably present in the cells. The addition of formaldehyde (FA), which easily permeates the cell wall and membrane, links the bait protein to its potential interaction partners.
The bait protein together with its potential interaction partners can then be isolated using a Strep-Tactin sepharose column by eluting with desthiobiotin- or biotin-containing buffer.
The cross-links between the bait protein and the potential interaction partner can be cleaved by heating the samples in Laemmli buffer. Finally, the co-purified interaction partner(s) can be separated by [http://www.ruf.rice.edu/~bioslabs/studies/sds-page/gellab2.html (SDS PAGE)], identified in mass spectrometry and Western Blotting.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 1
  Illegal suffix found in sequence at 1390
  Illegal EcoRI site found at 74
  Illegal PstI site found at 1232
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1
  Illegal EcoRI site found at 74
  Illegal NheI site found at 24
  Illegal NheI site found at 192
  Illegal SpeI site found at 1391
  Illegal PstI site found at 1232
  Illegal PstI site found at 1405
  Illegal NotI site found at 7
  Illegal NotI site found at 1398
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1
  Illegal EcoRI site found at 74
  Illegal BamHI site found at 95
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 1
  Illegal suffix found in sequence at 1391
  Illegal EcoRI site found at 74
  Illegal PstI site found at 1232
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 1
  Illegal EcoRI site found at 74
  Illegal XbaI site found at 16
  Illegal SpeI site found at 1391
  Illegal PstI site found at 1232
  Illegal PstI site found at 1405
  Illegal NgoMIV site found at 517
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 60