Difference between revisions of "Part:BBa K3098008"
 
(One intermediate revision by one other user not shown)
Line 5: Line 5:
 
In order to purify the PgD5 produced by E.Coli and increase its solubility, a GST tag is added at the N-terminal of the sequence
 
In order to purify the PgD5 produced by E.Coli and increase its solubility, a GST tag is added at the N-terminal of the sequence
 
.  
 
.  
 +
 +
===usage and biology===
 
GST tag can help the peptides dissolve in cytoplasm to help the expression of the peptide.During purification, GST tag can bind with the corresponding column to keep the target protein when the impurities leave. After washing, PreScission Protease (also have GST tag) will be added to cut the GST tag from the target protein, in this way, we'll be able to collect pure solution of the target protein without adding any more impurities.
 
GST tag can help the peptides dissolve in cytoplasm to help the expression of the peptide.During purification, GST tag can bind with the corresponding column to keep the target protein when the impurities leave. After washing, PreScission Protease (also have GST tag) will be added to cut the GST tag from the target protein, in this way, we'll be able to collect pure solution of the target protein without adding any more impurities.
 +
===WHU-China 2019===
 +
The expression quantity of PgD5 is so low that it could not be purified well. To enhance the expression quantity, we add 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 is the result.
 +
 +
<center>
 +
https://static.igem.org/mediawiki/parts/thumb/2/20/T-WHU-China-GST-PgD5-1.png/634px-T-WHU-China-GST-PgD5-1.png
 +
</center>
 +
The GST-Tag can be cleaved by PreScission Protease.
 +
We use the PPase cleave the fusion protein in cleavage buffer hung in 4℃ for 16h and condense the cleavage sample by PEG-20000. The blow is the result.
  
−
<!-- -->
+
<center>
 +
https://static.igem.org/mediawiki/parts/thumb/0/0e/Amp4.png/609px-Amp4.png
 +
</center>
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3098008 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3098008 SequenceAndFeatures</partinfo>

Latest revision as of 16:58, 21 October 2019


GST+PgD5

In order to purify the PgD5 produced by E.Coli and increase its solubility, a GST tag is added at the N-terminal of the sequence .

usage and biology

GST tag can help the peptides dissolve in cytoplasm to help the expression of the peptide.During purification, GST tag can bind with the corresponding column to keep the target protein when the impurities leave. After washing, PreScission Protease (also have GST tag) will be added to cut the GST tag from the target protein, in this way, we'll be able to collect pure solution of the target protein without adding any more impurities.

WHU-China 2019

The expression quantity of PgD5 is so low that it could not be purified well. To enhance the expression quantity, we add 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 is the result.

634px-T-WHU-China-GST-PgD5-1.png

The GST-Tag can be cleaved by PreScission Protease. We use the PPase cleave the fusion protein in cleavage buffer hung in 4℃ for 16h and condense the cleavage sample by PEG-20000. The blow is the result.

609px-Amp4.png

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