Difference between revisions of "Part:BBa K3098006"

Revision as of 15:31, 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. 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.

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

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 below was 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 below was the result.

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 below was the result.

Sequence and Features