Difference between revisions of "Part:BBa K3098006"

Latest revision as of 16:53, 21 October 2019

PgD5

The part contains the coding sequence for the antimicrobial peptide PgD5, which shows the ability to inhibit the growth of some filamentous fungi.

The tertiary structure of PgD5 has been shown here. For more information about the antimicrobial peptide, you can take the paper we have read as a reference:Identification of defensin-encoding genes of Picea glauca: characterization of PgD5, a conserved spruce defensin with strong antifungal activity

Usage and Biology

Some research about the peptide suggests that this kind of effect comes into being by disrupting the membrane structure of the cell. The expression of PgD5 in E.coli has been done in the research in order to get its purified solution, and a strong inhibition toward some kinds of filamentous molds has been proved in experiment. For the reasons above, we chose this special peptide in our project as a defender of the silk relics.

WHU-China 2019

At the beginning of the experiment, we expressed the PgD5 on the pET-28a(+) backbone, and used His-Tag at N-terminal to purify the protein. We did the Tris-Tricine SDS-PAGE electrophoresis and western-blot to detect the purification. The result was not encouraging. The following is the result of western-blot.

In the figure, we found that the expression quantity of PgD5 was so low that it could not be purified well. To enhance the expression quantity, we changed the backbone, from pET-28a(+) to pGEX6P-1. It had a GST-Tag at N-terminal. The GST-Tag could increase the solubility of fusion protein and avoid the inclusion body. We induced the expression of GST-PgD5 fusion protein and purified it by GST affinity column and eluted the fusion protein by GSH. We did SDS-PAGE electrophoresis, the following is the result.

We used the PPase to cleave the fusion protein in cleavage buffer (hung in 4℃ for 16h). We did the SDS-PAGE electrophoresis.

It was strange that, after cleavage, there was only the protein about 26kDa (may be GST) without the PgD5 in the SDS-PAGE gel. After repeated the experiment many times and analyzed the process, we speculated that we had succeeded in the cleavage but the concentration of PgD5 was too low to be stained by the Coomassie blue. As a substitution, we did silver staining. It proved our conjecture, so we condensed the cleavage sample by PEG-20000. The SDS-PAGE gel showed that we got the PgD5 successfully!

The next work was to test whether the PgD5 could inhibit the mold. We used the solvent (cleavage buffer in purification, pH~7.0) as the negative control, 10% NaOH as positive control and Aspergillus japonicus as the test object. To test the function of antimicrobial peptides in various pH environments, we adjusted the pH of the PDA medium (pH~5.5) to pH~7.5 and pH~8.0. The results indicates that PgD5 has a strong antifungal effect under pH~7.5 and pH~8.0.

Sequence and Features