Part:BBa_K3185000

SPYtag inserted 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 a biological polymer. We put Spytag inside TmEncapsulin because we used SpyTag/SpyCatcher system to bind it to other parts[2]. Also, this has three tag and cleavage 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 antibodies. 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 it like that[3].

We put it between the BamHI site and the Ndel site on pET11-a. We used BL21 (DE3) for gene expression. We used the Ni-NTA Agarose for purification. After that, we confirmed the molecular weight of SpyCatcher inserted TmEncapusulin 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 597
  
• 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 37^oC shaking at 140 rpm to an OD600 of 0.5, verifying via a spectrophotometer.

• Protein was expressed in 0.1mM IPTG for 2hours.

References

[1]Structural basis of enzyme encapsulation into a bacterial nano compartment
[2]Programmable polyproteins built using twin peptide superglues