Part:BBa_K243022

DsbA-Strep-DigA-Split Linker-Fok_a

This combination uses the benefits of a Streptavidin tag for purification. It is also linked with a DigoxigeninA tag DigA. The Split Linker connects the parts and adds additional space between them to guarantee the independent function of DigA tag and the protein domain Fok_a. The DsbA tag is used to transfer the produced protein into the periplasm.

Usage and Biology

This composite part is one part of our universal endonuclease and it needs another composite part like BBa_K243010 to build a functional heterodimer. The DigA tag guides the part to the DNA which is hybridized with a Digoxigenin labeled oligonucleotide. The split linker creates a distance of 51bp between the DigA and the linked Fok_a protein domain. The StrepTag serves as a purification tag for Streptavidin column purification.

We applied the Streptavidine tag to enable a simultaneous purification of constructs with a His tag. Strep tag also shows a higher affinity towards Strep-Tactin than His tag. For that the purification with Strep tag is more specific. The used DigoxigeninA tag allows the coupling to an fluorescein linked oligo. Emanating from our 3D modeling this combination of DigA tagged oligos and the construct containing the protein domain Fok_a is more efficient than the use of a combination of FluA tagged oligos with a construct containing Fok_a. To avoid interactions between the DigA tag with the connected protein domain Fok_a we applied the Split Linker. The Linker itself has no influence on the connected parts. We decided to use the Split Linker for this construct to get a longer distance between DigA tag and Fok_a to guarantee the independent function of both parts. This Linker is an improved part of the team Freiburg08 and it is a suitable linker for fusion proteins. The properties of the split linker are a good compromise between the stability and the distance of the connection between protein domain Fok_a and the anticalin tag. The additional linkage of the construct with DsbA allows the export of the protein into the periplasm. The accumulation of the expressed protein in the periplasm can be used for the purification of the protein.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 338
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1186