Difference between revisions of "Part:BBa K3098009"
 
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In this part, GST tag helps the dissolution and purification of the peptide while Avitag provides a site for biotinylation as a linkage to the dCBM3a and BC. It has been proved by our experiments that these modifications on the peptide will not eliminate the antifungal function of the peptides after our corresponding treatment, so we'll be able to produce functional antimicrobial peptides with this precursor.
 
In this part, GST tag helps the dissolution and purification of the peptide while Avitag provides a site for biotinylation as a linkage to the dCBM3a and BC. It has been proved by our experiments that these modifications on the peptide will not eliminate the antifungal function of the peptides after our corresponding treatment, so we'll be able to produce functional antimicrobial peptides with this precursor.
 
===WHU-China 2019===
 
===WHU-China 2019===
−
Expression quantity of PgD5-Avitag is so low that it could not be purified well. To enhance the expression quantity,we add GST-Tag at N-terminal, it 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.
+
Expression quantity of PgD5-Avitag was so low that it could not be purified well. To enhance the expression quantity,we add GST-Tag at N-terminal, it 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.
 
<center>
 
<center>
 
https://static.igem.org/mediawiki/parts/f/f2/T-WHU-China-GST-PgD5-Avitag-1.png
 
https://static.igem.org/mediawiki/parts/f/f2/T-WHU-China-GST-PgD5-Avitag-1.png

Latest revision as of 17:03, 21 October 2019


GST+PgD5+Avitag

This part is the one we are hoping to use in the production of the functional PgD5.

usage and biology

In this part, GST tag helps the dissolution and purification of the peptide while Avitag provides a site for biotinylation as a linkage to the dCBM3a and BC. It has been proved by our experiments that these modifications on the peptide will not eliminate the antifungal function of the peptides after our corresponding treatment, so we'll be able to produce functional antimicrobial peptides with this precursor.

WHU-China 2019

Expression quantity of PgD5-Avitag was so low that it could not be purified well. To enhance the expression quantity,we add GST-Tag at N-terminal, it 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.

T-WHU-China-GST-PgD5-Avitag-1.png

The GST-Tag can be cleaved by PreScission Protease. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 688
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 784
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 85