Part:BBa_K2686002

Encapsulin protein with HexaHistidine insert

This is a BioBrick containing the sequence for Thermotoga maritima encapsulin, a bacterial protein nanocompartment which self assembles to form a 60-mer.

Usage and Biology

The part can be used to deliver cargo, both on the outer surface of the nanoparticle by fusing a peptide in between the 139/140 Amino Acids as well as the protein's C terminus. Cargo proteins can also be loaded inside the nano-cage using a tag binding to Encapsulin's interior surface. The protein is modified with an additonal amino acid sequence (GGGGGGHHHHHHGGGGG) between positions 43/44 granting it better stability and high heat resistance (Moon et al., 2014).

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
  

Results

The sequence was tested inside a a pet14 vector backbone inside an operon with a T7 promoter and a T7 terminator.

Self Assembly

To determine whether the protein cage is successfully expressed in a cell-free environment, multiple PAGE gels were run. The Encapsulin 60-mer has a mass of around 1.3 MDalton, which should reflect in a band very high up on the gels.

References

Moon, H., Lee, J., Min, J. and Kang, S. (2014). Developing Genetically Engineered Encapsulin Protein Cage Nanoparticles as a Targeted Delivery Nanoplatform. Biomacromolecules, 15(10), pp.3794-3801.