Difference between revisions of "Part:BBa K3185001"

Revision as of 13:48, 17 October 2019

Tm Encapsulin

Usage and Biology

TmEncapsulin is a protein found from Thermotoga maritima. A paper (Structural basis of enzyme encapsulation into a bacterial nanocompartment) says that it consists of 60 monomers and forms capsule, Virus-like particle(VLP). iGEM also treats it as a useful part (BBa_K192000).

We used TmEncapsulin as biological polymer, because it consists of 60 monomers. Also, this has three tag 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 the antibody. Third is a 6x-His tag inserted between the C-terminus of Encapsulin and 6x-His tag because, in a paper [Developing Genetically Engineered Encapsulin Protein Cage Nanoparticles as a Targeted Delivery Nanoplatform], 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 like that.

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 molecular weight of 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