Difference between revisions of "Part:BBa K3185000"
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− | We used TmEncapsulin as a biological polymer. We inserted SpyTag in TmEncapsulin. This enables TmEncapsulin to display different types of proteins on the surface of the protein capsule. (See Fig.2) (SpyCatcher:''<partinfo>BBa_K1159200</Partinfo>'', SpyTag:''<partinfo>BBa_K1159201</partinfo>'')[2]. SpyTag forms an isopeptide bond with SpyCatcher when they are mixed[4]. In previous research about TmEncap, it is showed peptides inserted after 138th amino acid in TmEncap can be exposed on the protein capsule as a loop[3]. Furthermore, “Author et.al” showed when SpyTag is inserted at the same position, SpyCatcher/SpyTag also forms a bond between SpyCatcher and SpyTag inserted TmEncap (SpyTmEnc)[5]. Also, this has three tag and cleavage 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. (''<partinfo>BBa_K2686002</partinfo>'') | + | We used TmEncapsulin as a biological polymer. We inserted SpyTag in TmEncapsulin. This enables TmEncapsulin to display different types of proteins on the surface of the protein capsule. (See Fig.2) (SpyCatcher:''<partinfo>BBa_K1159200</Partinfo>'', SpyTag:''<partinfo>BBa_K1159201</partinfo>'')[2]. SpyTag forms an isopeptide bond with SpyCatcher when they are mixed[4]. In previous research about TmEncap, it is showed peptides inserted after 138th amino acid in TmEncap can be exposed on the protein capsule as a loop[3]. Furthermore, “Author et.al” showed when SpyTag is inserted at the same position, SpyCatcher/SpyTag also forms a bond between SpyCatcher and SpyTag inserted TmEncap (SpyTmEnc)[5]. <br> |
+ | Also, this has three tag and cleavage 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. (''<partinfo>BBa_K2686002</partinfo>'') | ||
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Revision as of 09:43, 20 October 2019
SPYtag inserted 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).
We used TmEncapsulin as a biological polymer. We inserted SpyTag in TmEncapsulin. This enables TmEncapsulin to display different types of proteins on the surface of the protein capsule. (See Fig.2) (SpyCatcher:BBa_K1159200, SpyTag:BBa_K1159201)[2]. SpyTag forms an isopeptide bond with SpyCatcher when they are mixed[4]. In previous research about TmEncap, it is showed peptides inserted after 138th amino acid in TmEncap can be exposed on the protein capsule as a loop[3]. Furthermore, “Author et.al” showed when SpyTag is inserted at the same position, SpyCatcher/SpyTag also forms a bond between SpyCatcher and SpyTag inserted TmEncap (SpyTmEnc)[5].
Also, this has three tag and cleavage 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 the BamHI site and the Ndel site on pET11-a. We used BL21 (DE3) for gene expression. We used the Ni-NTA Agarose for purification. After that, we confirmed the molecular weight of SpyCatcher inserted TmEncapusulin by using SDS-PAGE.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 77
Illegal BglII site found at 597 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 426
Illegal SapI.rc site found at 457
Purification
Expression
- Cells were grown in 200ml LB media (100μg/ml Ampicillin) at 37oC 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
Result
![](/wiki/images/9/93/Peak191015.png)
TmEncapsulin expressed E. coli lysate and purified protein solution was loaded on 10%-60% sucrose linear gradient / 20 mM Tris 7.5, 50 mM NaCl, then centrifuged in 100,000g for 18 hours at 4℃ with SW41(Beckman). The solution was fractionated on a 96-well plate with BioComp. At the same time, 260nm absorption was measured.
![](/wiki/images/f/f6/191001_SPYt-SPYC_2.png)
3µL of SpyCatcher-Plastic-binding protein (SpyC-PBP) solution and 3µL of SpyTag inserted TmEncapsulin (SpyTmEnc) solution was mixed, then placed for 16h at room temperature. Then 6µL of 2x SDS sample buffer was added. 10µL of each sample was loaded. SDS-PAGE for 30min in 200V. The gel was CBB stained.
References
1 Putri, R.M., Allende-Ballestero, C., Luque, D., Klem, R., Rousou, K.A., Liu, A., Traulsen, C.H.H., Rurup, W.F., Koay, M.S.T., Castón, J.R., et al. (2017).
Structural Characterization of Native and Modified Encapsulins as Nanoplatforms for in Vitro Catalysis and Cellular Uptake.
ACS Nano 11, 12796–12804.
2 Veggiani, G., Nakamura, T., Brenner, M.D., Gayet, R. V., Yan, J., Robinson, C. V., and Howarth, M. (2016).
Programmable polyproteams built using twin peptide superglues.
Proc. Natl. Acad. Sci. U. S. A. 113, 1202–1207.
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.
4 Zakeri, B., Fierer, J.O., Celik, E., Chittock, E.C., Schwarz-Linek, U., Moy, V.T., and Howarth, M. (2012).
Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin.
Proc. Natl. Acad. Sci. U. S. A. 109.
5 Bae, Y., Kim, G.J., Kim, H., Park, S.G., Jung, H.S., and Kang, S. (2018).
Engineering Tunable Dual Functional Protein Cage Nanoparticles Using Bacterial Superglue.
Biomacromolecules 19, 2896–2904.