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Revision as of 14:00, 17 October 2019


sfGFP / Tm Encapsulin

Usage and Biology

TmEncapsulin is a protein found from Thermotoga Maritima. A paper (Structural basis of enzyme encapsulation into a bacterial nano compartment) says that it consists of 60 monomers and forms capsule, Virus-like particle(VLP). iGEM also treats it as a useful part (BBa_K192000). According to the paper, (Reference)TmEncapsulin can enclose proteins with cargo loading protein (CLP).

Moreover, we inserted CLP on the C-terminus of superfolder GFP (sfGFP) whose folding interval is shortened by improving natural GFP, and we inserted it to the upstream of TmEncapsulin. It is thought that we can confirm the end of construction by investigating fluorescence because sfGFP is enclosed inside a VLP when completing 60mer TmEncapsulin.

We used TmEncapsulin as a biological polymer because it consists of 60 monomers. Also, it 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 [Developing Genetically Engineered Encapsulin Protein Cage Nanoparticles as a Targeted Delivery Nanoplatform], 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 it like that.

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 the molecular weight of sfGFP-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 853
    Illegal BglII site found at 1268
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 13
    Illegal SapI.rc site found at 1202
    Illegal SapI.rc site found at 1233

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



References