Part:BBa_K3815002

Defensin1-Mxe GryA intein-PTlinker-ELK16

Description of this part

Targeted protein

This part is for the purfication of antimicrobial peptide, Defensin1. This is derived from Homo Sapiens. This can inhibit the growth of the gram-positive bacteria. In our experiment, we used it to kill the bacteria in the vase water.

Purification system

Fig1.The mechanism of ELK16

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 GyrA 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
  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]

Fig2. 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, 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 2, 6,and 10 are the result of Defensin1.
Defensin1 is 5713Da, so these date shows that we could confirm its production.

Reference

1.Fujiwara, S., Imai, J., Fujiwara, M., Yaeshima, T., Kawashima, T., and Kobayashi, K. (1990). A potent antibacterial protein in royal jelly. Purification and determination of the primary structure of royalisin. J. Biol. Chem. 265, 11333–11337.
2.https://parts.igem.org/Part:BBa_K1104301