Coding

Part:BBa_K3815007

Designed by: Hiroto Koga   Group: iGEM21_Kyoto   (2021-10-16)


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

Description of this part

Targeted protein

This part is for the purification of the antimicrobial peptide, Defensin1. This is derived from Homo sapiens. This can inhibit the growth of 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 GyrA intein, PT linker, and targeted protein. This is for the peptide intein tag purification. 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. This method saves time removing the tags compared to the method using only tags without intein.

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]

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 200μL 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 2 and 8 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.http://parts.igem.org/Part:BBa_K1104301

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