Part:BBa_K3185002

sfGFP / 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). According to the paper, (Reference)TmEncapsulin can enclose proteins with cargo loading protein (CLP) [2].

Moreover, we inserted CLP on the C-terminus of superfolder GFP (sfGFP, BBa_I746916) 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 6x-His tag placed in the C-terminus of TmEncapsulin for protein purification by using Ni-NTA beads. However, in a paper, Ni-NTA beads cannot bind to 6x-His tag added in C-terminus because it doesn’t display enough to the surface of the protein capsule [3]. To solve this problem, we inserted second tag. Second is HAtag inserted between TmEncapsulin and 6x-His tag in expectation of C-terminus to display on the surface of the capsule. Third is 6x-His-tag and linker inserted between #43 and #44 amino acids of native encapsulin for improving heat-resistance of TmEncapsulin. To design third one, we refered BBa_K2686002 of iGEM EPFL 2018 and the same paper.(BBa_K2686002)

We put it between BamHI site and Ndel site on pET11-a. The expression plasmids were introduced into BL21(DE3) and expressed by T7 promoter/ T7 RNAP system. Ni-NTA agarose was used for the purification.

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

Fig.1 SDS-PAGE of imidazole elutes, CBB stained

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.

Purification

1. E. coli which expressed this part were lysed with sonification.
2. Proteins are purified from lysate with Ni-NTA agarose(QIAGEN).
3. Imidazole eluates were visualized and confirmed by SDS-PAGE followed by CBB staining.

This purification method works. As shown in Fig.1, the protein successfully purified.

Result

TmEnencapsulin has 6x-His tag for purification but sfGFP doesn't have it, so if they are purified separately, we can't get sfGFP. However, as shown in Fig.1, there are two bands in each lanes, which indicates that TmEnencapsulin loads sfGFP.

References

1 Sutter, M., Boehringer, D., Gutmann, S., Günther, S., Prangishvili, D., Loessner, M.J., Stetter, K.O., Weber-Ban, E., and Ban, N. (2008).
Structural basis of enzyme encapsulation into a bacterial nanocompartment.
Nat. Struct. Mol. Biol. 15, 939–947.

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

3 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.