Part:BBa_K2686006

Encapsulin protein with HexaHistidine insert, OT1 peptide at C terminus and a sfGFP-tag protein.

This Composite part consists of an encapsulin protein BBa_K2686001 with a HexaHistidine linker BBa_K2686002 and an OT1 peptide fused to the C terminus BBa_K2686000, followed by a RBS and a sfGFP protein fused with a 15 amino acid long peptide tag. This peptide tag allows the sfGFP to bind to the encapsulin's interior surface, thus allowing the loading of sfGFP in the Encapsulin multimer (Cassidy-Armstutz et al., 2016). This would allow having a fluorescent Encapsulin protein.

This part is derived from BBa_K2686005.

Purification

After having tested a variety of purification procedures, heat purification at 70C for 20 minutes followed by cooling on ice for 15 minutes and a subsequent centrifugation at 12000g for 10 minutes was found to be the most efficient way of isolating the encapsulin (encapsulin ends up in supernatant).

Assembly

The self assembly of the encapsulin 60-mer was first examined using SDS PAGE, where a band around 30.71kDa band is expected to form. Additionally due to Encapsulin's exceptional heat stability the 1.98MDa complex also appears on the gel after SDS denaturation.

Native PAGE gel of sfGFP, HexaHistidine-OT1 Encapsulin and HexaHistidine-OT1 Encapsulin sfGFP-tag. On the left is the fluorescent image of the gel and o the right is the gel after Coomassie staining. Before (B) heat purification procedure outlined in Purification section, Supernatant (S) after the heat purification procedure. Stars ★ show the bands for monomers of HexaHistidine-OT1 Encapsulin (BBa_K2686000,BBa_K2686006) with sfGFP-tag bound to them, increasing the molecular weight of the complex when compared to sfGFP alone. Triangles ▲ signify a 60-mer band to the right. From left to right: (1-2) Negative control (cell-free TX-TL expression without DNA), (2-4) HexaHistidine-OT1 Encapsulin (BBa_K2686000), showing no fluorescence on the left gel, has a band for 60-mer on stained gel (▲). (5-6) sfGFP protein, the fluorescent bands can be easily seen as large smears on the left. (7-8) HexaHistidine-OT1 Encapsulin and sfGFP-tag (BBa_K2686006) shown by a ★. The difference in height between the bands of sfGFP compared to the ★ is striking and suggests that the sfGFP-tag binds to the HexaHistidine-OT1 Encapsulin monomers. In addition there seems to be a small amount of 60-mer indicated by ▲. (9-10) Encapsulin (BBa_K2686001) does assemble to form the 60-mer ▲ as seen on the stained gel. (11) HexaHistidine-OT1 Encapsulin and sfGFP-tag (BBa_K2686006), on the left the presumed ★ protein dimer is seen to be higher than the sfGFP in lane 12, no particular bands can be identified on the right. (12) sfGFP, fluorescent band is seen to be lower than for lane 11.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 902
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 77
  Illegal BglII site found at 492
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 902
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 902
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 426
  Illegal SapI.rc site found at 457
  Illegal SapI.rc site found at 955
  

References

Cassidy-Amstutz, C., Oltrogge, L., Going, C., Lee, A., Teng, P., Quintanilla, D., East-Seletsky, A., Williams, E. and Savage, D. (2016). Identification of a Minimal Peptide Tag for in Vivo and in Vitro Loading of Encapsulin. Biochemistry, 55(24), pp.3461-3468.