Difference between revisions of "Part:BBa K3185002"

Revision as of 08:05, 20 October 2019

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).

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[2]. 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. 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

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.

SDS-PAGE

alt text

Result

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 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.