Part:BBa_K4440001

preproSP-4B4-AGA2-Tyr1-3HA

These five protein modules (SP, 4B4 peptide, Aga2, Tyr1, 3HA tag) are fused together by flexible linkers. Signal peptide from the alpha factor directs the engineered protein to the secretory pathway. Once secreted, Aga2 anchors the protein to the cell wall. 4B4 peptide is responsible for the binding of the synthesized melanin to accumulate it on the cell surface (Ballard et al., 2011). 3HA tag is used to later test the presence of the enzyme on the cell surface.

Usage and Biology

This part encodes a fusion protein consisting of multiple parts: SP - synthetic α-factor prepro signal-peptide, which is used to guide the protein to the cell surface, 4B4 - sequence encoding for a peptide that selectively binds melanin, Tyr1 - gene encoding for B. megaterium tyrosinase, 3HA tag - Human influenza hemagglutinin protein tag and G4S, G2S linkers - flexible poly-Glycine-Serine linkers between different protein fusion domains (Figure 1).

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Figure 1. A scheme representing Aga2-Tyr1, a fusion protein of five domains to drive cell-wall-directed melanin synthesis and accumulation.

The five protein modules in this sequence (SP, 4B4 peptide, Aga2, Tyr1, 3HA tag) are fused together by flexible linkers. Signal peptide from the α-factor directs the engineered protein to the secretory pathway. Once secreted, Aga2 anchors the protein to the cell wall. 4B4 peptide is responsible for the binding of the synthesized melanin to accumulate it on the cell surface, creating a protective shield on the cell surface (Ballard et al., 2011). This part mediates melanin production outside the cell, possibly decreasing the negative effects of synthesis.

Protein domains are often modular, but assembling five protein domains to form a novel protein always carries risks of poor protein expression, degradation, or proteolysis. To test the Aga2-Tyr1 fusion protein expression, we collected samples before (0 h) and 0.5 h, 1.5 h, and 24 h after galactose induction. We analyzed the samples by western blotting using antibodies against the HA epitope tag. The experiment showed that Aga2-Tyr1 is expressed at a comparable level to another protein used as a positive control and that the tyrosinase expression is dependent on galactose (Figure 2).

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Figure 2. Western blot confirms galactose-induced expression of Aga2-Tyr1-3HA. Samples were collected at the indicated time points since galactose addition. Western blot using α-3HA antibody is shown.

After confirming that our protein is expressed we aimed to test its ability to promote melanin production. For this, we constructed a yeast strain with this sequence integrated into its genome. The resulting strain was used for several analyses.

First, we checked if the cells will change color as the accumulation of melanin leads to cell pigmentation. As can be seen from Figure 3, the cells where Aga2-Tyr1 was expressed indeed changed their color.

Figure 3. Color change in yeast cells upon expression of Aga2-Tyr1.

References:

Ballard, B., Jiang, Z., Soll, C. E., Revskaya, E., Cutler, C. S., Dadachova, E., & Francesconi, L. C. (2011). In vitro and in vivo evaluation of melanin-binding decapeptide 4B4 radiolabeled with 177Lu, 166Ho, and 153Sm radiolanthanides for the purpose of targeted radionuclide therapy of melanoma. Cancer Biotherapy & Radiopharmaceuticals, 26(5), 547–556. https://doi.org/10.1089/CBR.2011.0954

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]