Part:BBa_K3815001

CecropinA-Mxe GryA intein-PT-linker-ELK16

Usage and Biology

Targeted protein

This part is for the purfication of antimicrobial peptide, CecropinA. This is derived from Hyalophora cecropia. The antimicrobial peptide has the 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

In order to purify the peptide, we adopted ELK16 system that the past iGEM teams had not used. This part is composed of ELK16, Mxe Gyr intein, and PT linker. When this fused protein is produced, ELK16 self-assembles and precipitates. After that, taking this aggregate and adding DTT to it, the N terminal of Mxe GyrA intein is cut. Then, we can get the targeted protein.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 814
  
• 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 37^oC shaking at 180 rpm.
• when the OD exceeded 0.35, IPTG 2ml was added to induce the peptide expression.

Purification

1.When this fused protein were produced, it self-assembled and precipitated
2.The aggregate was collected by centrifugation.
3.Adding 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 1, 5,and 9 are the result of CecropinA.
CecropinA is 4051Da, so these date shows that we could not purify it sufficiently.