Part:BBa_K404222

[AAV2]-Rep-VP123_P5-TATAless (ViralBrick-453-His-Tag)

Usage and Biology

Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein.
The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(Smith et al. 1988), (Hoffmann & Roeder 1991)
Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. Koerber et al. have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC (Koerber et al. 2007). For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing for an easy insertion into the 453 and/or 587 loop. If the modified capsid bearing a His-tag is being cotransfected with a wild type capsid for the production of mosaic viruses, IMAC helps to not only purify the produced viral particles but also to enrich particles which actually contain the modified proteins.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 3635
  Illegal XhoI site found at 1913
  Illegal XhoI site found at 2099
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 4161
  Illegal BsaI site found at 4343
  Illegal BsaI site found at 4380
  Illegal SapI site found at 3048