Part:BBa_K4035001

CUP1 fused to Aga2 and tagged with a V5 epitope

This protein is made of the copper metallotionein CUP1 (BBa_M45090) fused to the A-agglutinin-binding subunit Aga2 (BBa_K416000) at its N-terminal and to the V5 tag at its C-terminal.

Usage and Biology

Copper metallotionein 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 leading to the presence of CUP1 on the outter membrane of the cell. This fusion protein also contained a V5 tag in order to check its expression by Western Blot and Immunostaining. The expression was under the control of the Gal1 promoter, so that the protein was expressed only in the presence of galactose.

Characterization

Expression of the protein in the recombinant yeast :

Figure 1 : Western Blot results

The first two lines, EBY100 and pCTcon2V5 without galactose which are, respectively, wild type yeast and uninduced transformed yeast with the backbone plasmid, serve as negative controls, and show no presence of the V5 tag, as we expected. As the plasmid contains a Gal1 promoter, the system can only be expressed in the presence of galactose. The third line, pCTcon2V5 + galactose is the induced transformed yeast with the backbone plasmid and shows expression of the V5 tag which proves that our system is expressed in the transformed yeast when induced with galactose. The fourth line is our transformed yeast with the recombinant plasmid pCTcon2-CUP1-V5 and also shows expression of the system. We can see that we have two lines of approximately 20 to 25 kDa which is the size of the fusion protein Aga2-CUP1-V5. The smaller line could be a truncated version of the protein as we later identified a second in-frame start codon in the DNA sequence. Unfortunately the truncated version seems to be the most expressed one, corresponding to the signal intensity.

Figure 2 : Immunostaining of the yeast transformed with the pCTcon2-CUP1-V5 plasmid

The blue dots represent the nucleus staining with DAPI and the green disks are the recombinant yeats cells expressing CUP1 at their surface. Due to the more intense circle we can clearly see that our system is expressed on the membrane of the protein. As these are non permeabilized cells, the antibodies bind only the extracellular proteins. We can also remark that not all the cells are expressing the system. This mainly due to...

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 319
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 376
  Illegal PstI site found at 319
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 562
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 319
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 319
  
• 1000
  COMPATIBLE WITH RFC[1000]