Part:BBa_K4035000

Expression of CUP1 on the extracellular membrane of S. cerevisiae

This part is the expression system of the CUP1 fused to Aga2 and tagged with a V5 epitope (BBa_K4035001). It has been made by inserting the endegenous CUP1 gene into the pCTcon2V5 plasmid(1) and is composed of the Gal1 promoter (BBa_J63006), the A-agglutinin-binding subunit Aga2 (BBa_K416000) linked to the CUP1 copper metallotionein protein 1 (BBa_M45090) and the V5 tag (BBa_T2020).

Usage and Biology

Copper metallothionein CUP1 (BBa_M45090) is a protein responsible for copper binding protein in the yeast Saccharomyces cerevisiae. Normally expressed intracellularly, CUP1 was fused to the A-agglutinin-binding subunit Aga2 (BBa_K416000) that attaches to the yeast cell wall through disulfide bonds to Aga1p. This leads to the presence of CUP1 on the outer membrane of the cell. The goal behind it was to improve the endogenous bio-accumulation property of wild type yeast by allowing it to bind more copper ions on its external surface.

This fusion protein also contains a V5 tag (BBa_T2020) in order to check its expression by Western Blot and Immunocytochemistry. The expression is under the control of the Gal1 promoter (BBa_J63006), so that the protein is expressed only in the presence of galactose.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 150
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

The characterization of this part can be found on the Part:BBa K4035001 page that is describing the fusion protein Aga2-CUP1-V5.