Difference between revisions of "Part:BBa K3815006"
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<h3><font size="4.5">Purification </font></h3>
 
<h3><font size="4.5">Purification </font></h3>
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1.When this fused protein were produced, it self-assembled and precipitated<br>
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1.When this fused protein were produced, it was recovered by Ni chromatography.<br>
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2.The aggregate was collected by centrifugation.<br>
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2.Adding DTT to it, the targeted protein was cut out by the cleavage of intein.<br>
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3.Adding 40mM DTT to this aggregate, the targeted protein was cut out by the cleavage of intein.<br>
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3.SDSPAGE was performed in order to confirm the presence of it.
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4.SDSPAGE was performed in order to confirm the presence of it.
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Revision as of 12:04, 20 October 2021


CecropineA-Mxe GryA intein-PT-linker-His tag

Descriotion of this part

Targeted protein

This part is for the purfication of antimicrobial peptide, CecropinA. This is derived from Hyalophora cecropia. This can inhibit the growth of both gram-negative bacteria and gram-positive bacteria. In our experiment, we used it to kill the bacteria in the vase water.

Purification system

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 67
    Illegal AgeI site found at 346
  • 1000
    COMPATIBLE WITH RFC[1000]

Purification

Fig1. SDS-PAGE of purified peptide

Expression

  • Cells were grown in 1000ml LB media at 37oC shaking at 180 rpm.
  • when the OD exceeded 0.35, 1 M IPTG 2ml was added to induce the peptide expression.
  • Incubate at 30℃ at 180rpm for 6 hours after adding IPTG.

Purification

1.When this fused protein were produced, it was recovered by Ni chromatography.
2.Adding DTT to it, the targeted protein was cut out by the cleavage of intein.
3.SDSPAGE was performed in order to confirm the presence of it.

Fig1 shows the result of SDS-PAGE. The lane 1, 5,and 9 are the result of CecropinA.
CecropinA is 4051Da, so these date shows that we could not confirm CecA production.