Part:BBa_K3815007

Defensin1-Mxe GryA intein-PT-linker-His tag

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

This part is composed of His tag, Mxe Gyr intein, PT linker, and targeted protein. This is for the peptide purification used His tag. Producing peptide with His tag, it is recovered by Ni chromatography. After that, adding DTT to it, the N terminal of Mxe GyrA intein is cut. Then, we get the targeted protein.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 249
  Illegal SpeI site found at 573
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 249
  Illegal SpeI site found at 573
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 249
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 249
  Illegal SpeI site found at 573
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 249
  Illegal SpeI site found at 573
  Illegal NgoMIV site found at 682
  
• 1000
  COMPATIBLE WITH RFC[1000]

Expression

• Cells were grown in 1000ml LB media at 37^oC 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 self-assembled and precipitated
2.The aggregate was collected by centrifugation.
3.Adding 40mM DTT to this aggregate, the targeted protein was cut out by the cleavage of intein.
4.SDSPAGE was performed in order to confirm the presence of it.

Fig1 shows the result of SDS-PAGE. The lane 4, 8,and 12 are the result of NOP1.
NOP1 is 1132Da, so these date shows that we could not confirm its production.