Part:BBa_K1980000

TAT Copper Storage Protein 1

Copper Storage protein 1 (Csp1) is a tetrameric copper storage protein found in the periplasm of Methylosinus trichosporium OB3b. We investigated whether this part could act as a copper chelator when expressed in E. coli. We modified the protein by adding a TAT signal peptide from the E. coli enzyme CueO in place of the native TAT sequence and added a C terminal his tag to aid purification.

Usage and Biology

Our project aimed to detect and chelate dietary copper as a treatment for Wilson's Disease, a copper accumulation disorder. We decided that the ideal copper chelation protein would have these properties:

• Should be able to bind multiple copper ions per peptide to increase the efficient use of cell resources.
• They should be from the prokaryotic domain because eukaryotic proteins can have expression issues in Escherichia coli.
• As E. coli naturally deals with copper toxicity by binding copper in the periplasm then exporting it, periplasmic proteins may reduce toxicity to the host.

Using these criteria we found two copper chelators that we thought would be useful. We designed gBlocks, codon optimised for E. coli, containing these parts both alone and linked to super-folder <a data-toggle="popover1" data-trigger="hover" title="Green Fluorescent Protein" data-content="A protein that emits green light when illuminated with blue to UV light">GFP</a>. This form of the fluorescent protein was used because the standard GFP doesn’t fold particularly well in the periplasm where one of our chelators is intended to travel to. The proteins, both alone and attached to sfGFP, were ordered with C terminal <a data-toggle="popover1" data-trigger="hover" placement:"top" title="His Tag" data-content="Six sequential Histidine residues that bind to NTA columns"> hexa-histidine tag</a> so we could purify them. A concern was raised that the His tag would also weakly bind copper potentially affecting the results. However we decided that increasing the copper-binding would only improve the proteins' intended function.

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]