Coding

Part:BBa_K3185001

Designed by: Masahiro Sakono   Group: iGEM19_Kyoto   (2019-10-04)
Revision as of 06:42, 19 October 2019 by Daidai (Talk | contribs) (→‎Purification)


Tm Encapsulin

Usage and Biology

TmEncapsulin is a protein found from Thermotoga maritima. A paper says that it consists of 60 monomers and forms capsule, Virus-like particle(VLP)[1]. iGEM also treats it as a useful part (BBa_K192000).

We used TmEncapsulin as biological polymer, because it consists of 60 monomers. Also, this has three tag sites. First is 6×His-tag inserted in the C-terminus of TmEncapsulin for protein purification. Second is HA-tag inserted between TmEncapsulin and 6x-His tag to detect it by using the antibody. Third is a 6x-His tag inserted between the C-terminus of Encapsulin and 6x-His tag because, in a paper, it is said that 6x-His tag inserted in the C-terminus of Encapsulin is not presented on the surface of Encapsulin well, so it can’t bind to Ni-NTA Agarose beads .In the same paper, it is also said that heat-resistance is improved when inserting 6x-His tag and linker between #43 and #44 amino acids of native encapsulin, so we designed like that[2].

We put it between BamHI site and Ndel site on pET11-a. We used BL21 (DE3) for gene expression. We used Ni-NTA Agarose beads for purification. After that, we confirmed molecular weight of TmEncapsulin by using SDS-PAGE.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 77
    Illegal BglII site found at 492
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 426
    Illegal SapI.rc site found at 457

Purification


Expression

  • Cells were grown in 200ml LB media (100μg/ml Ampicillin) at 37oC shaking at 140 rpm to an OD600 of 0.5, verifying via a spectrophotometer.
  • Protein was expressed in 0.1mM IPTG for 2hours.

SDS-PAGE



alt text

References

1 Lukarska, M., Fournier, G., Pflug, A., Resa-Infante, P., Reich, S., Naffakh, N., and Cusack, S. (2017).
Structural basis of an essential interaction between influenza polymerase and Pol II CTD.
Nature 541, 117–121.

2 Moon, H., Lee, J., Min, J., and Kang, S. (2014).
Developing genetically engineered encapsulin protein cage nanoparticles as a targeted delivery nanoplatform.
Biomacromolecules 15, 3794–3801.

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