Team:Kyoto/Improve Part
Improve Part
Overview
We improved “Encapsulin protein with HexaHistidine insert” (BBa_K2686002) to “Spytag inserted TmEncapsulin” (BBa_K3185000) . We introduced two modifications to the existing part. First, we improved purification efficiency by adding 6x-His tag to C-terminus. Second, we enabled displaying different types of proteins on the surface of protein capsule by inserting SpyTag.
1st improve point: Improve purification efficiency by adding 6x-His tag to C-terminus
TmEncapsulin is a protein found from Thermotoga maritima. TmEncapsulin assembles sphere-like protein capsule as 60mer. This protein capsule could be applied to different situations, and ways to overexpress with E-coli and purify have been researched for a long time.
Encapsulin on existing basic parts page promoted thermotolerance of native TmEncapsulin by introducing 6x-His tag between 43rd and 44th amino acid. After expressing this by E.coli cell-free system, we can purify encapsulin by collecting supernatant after heat treatment and centrifugation.
We added 6x-His tag to the C-terminus of an existing part. This is due to make them available to protein purify by using Ni-NTA beads. However, in a paper suggested, 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 [1]. Here, we placed HAtag between TmEncapsulin and 6x-His tag in expectation of C-terminus to display enough on the surface of the capsule. We compared E.coli lysate before purification with a heat-treated product and Ni-NTA purified product with SDS-PAGE.
Shown in the Fig.1 below, we succeeded in purifying Encapuslin by using Ni-NTA beads. You can see that the product purified by Ni-NTA has a darker band. Also, we quantified band intensity, then calculated purified protein concentration. As shown in Fig.2, we were able to purify with five times as much concentration than the existing part by using Ni-NTA. This improvement means we can purify encapsulin more easily with high concentration.
Fig.1 TmEncapsulin was purified more efficiently with Ni-NTA
TmEncapsulin expressed E.coli was sonicated lysed with sonication. In the “Lysate” lane, the lysate was loaded. In the “Heat” lane, heat-treated supernatant was loaded. In the “Ni-NTA” lane, the protein was purified with Ni-NTA beads from the lysate, then loaded.
TmEncapsulin expressed E.coli was sonicated lysed with sonication. In the “Lysate” lane, the lysate was loaded. In the “Heat” lane, heat-treated supernatant was loaded. In the “Ni-NTA” lane, the protein was purified with Ni-NTA beads from the lysate, then loaded.
Fig.2 Ni-NTA beads purification got about 5 times concentrated TmEncapsulin
SDS-PAGE that showed in Fig.1 was triplicated, then band intensities were quantified with ImageJ. Then each protein concentration of the solution was calculated.
SDS-PAGE that showed in Fig.1 was triplicated, then band intensities were quantified with ImageJ. Then each protein concentration of the solution was calculated.
2nd improve point: Protein display through SpyCatcher/SpyTag system
There are many possible usages of Encapsulin. One is protein displaying on the surface of the capsule. For example, iGEM EPFL 2018 which created the existing part also created Encapsulin with HexaHistidine insert and C-terminal OT1 (BBa_K2686000) which are antigens of “OT1”. This modification aims to display antigen to immune cells effectively.
In order to make protein displaying easier, we inserted SpyTag in TmEncapsulin. SpyTag forms an isopeptide bond with SpyCatcher when they are mixed [2]. In previous research about TmEncap, it is shown peptides inserted after 138th amino acid in TmEncap can be exposed on the protein capsule as a loop [1]. Furthermore, Bae et al. showed when SpyTag is inserted at the same position, SpyCatcher/SpyTag also forms a bond between SpyCatcher(SpyC) and SpyTag inserted TmEncap (SpyTmEnc) [3]. This modification enables us to display different types of protein on SpyTmEnc through SpyCatcher. (See Fig.3)
Fig.3 Visual description of our second improvement
Different types of SpyCatcher fused protein can be displayed on SpyTmEnc.
Different types of SpyCatcher fused protein can be displayed on SpyTmEnc.
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. CBB-stained.
References
1 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. 2 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. 3 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.
Developing genetically engineered encapsulin protein cage nanoparticles as a targeted delivery nanoplatform.
Biomacromolecules 15, 3794–3801. 2 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. 3 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.
