Team:Kyoto/Design

Design
Introduction
Our device, "SONOBE", has three components.
  • Part.1 Plastic-binding protein
  • Part.2 Encapsulin
  • Part.3 SpyCatcher / Spytag
With these, we built "SONOBE" that aggregates microfibers. Here, we show the detail and research background of each component protein.
Part.1 Plastic-Binding Protein
As shown in the list of protein below, several papers reported proteins that bind to PET film, nanoparticle or other kinds of plastics. We prepared several proteins for PET fiber binding, then compared them with each other. BaCBM2, CenA, TA2, LCI KR-2, Hydrophobin, PETase, and engineered PETase were assessed in our project. All plastic-binding proteins are designed in the form of “SpyCatcher->plastic-binding protein”.
  • BaCBM2：
    BaCBM2 is a Carbohydrate-Binding Module (CBM) from Bacillus anthracis. CBM is often found in carbohydrate related enzymes. It can bind to not only highly crystallized cellulose but also to PET because it has a binding site formed by aromatic amino acids [1]. In another paper, it is shown that this protein binds to PET film and nanoparticle [2].
  • CenA :
    CBM-CenA is a Carbohydrate-binding Module from cellulase of Cellulomonas fimi. Originally, it has binding affinity to cellulose, but its binding ability to PET was enhanced by point mutation [3]. In the paper, this protein binds to PET fiber and PET filter.
  • TA2：
    Tachystation A2(TA2) is a protein from Tachypleus tridentatus [4]. The paper shows it binds to polyurethane (PU)[5].
  • LCI KR-2：
    LCI is a protein from Bacillus subtilis. The paper shows that it can bind to polypropylene(PP)[6]. Another paper shows the improved variant, LCI KR-2(Y29R and G35R; variant KR-2)[7]. Its affinity is 5.4±0.5 times stronger than natural LCI.
  • Hydrophobin：
    Hydrophobin is a protein from Bacillus subtilis. In this paper, it shows that they self-assemble and get hydrophobic region [8]. iGEM OLS_Canmore_Canada 2018 team used this protein as PET binding protein. Because PET is hydrophobic, hydrophobin might have hydrophilic interaction with PET.
  • PETase：
    PETase is a protein found from Ideonella sakaiensis. A paper shows that PETase has PET degradation activity [9].
  • Engineered PETase：
    Engineered PETase is a protein from Ideonella sakaiensis. In a paper, the binding activity and the degradation activity of the PETase enzyme were both improved by introducing mutations [10].
Part.2 Encapsulin
Encapsulin is a protein found in some bacteria and archaea species. This protein spontaneously forms a capsule-shaped polymer. The function of the protein capsule varies from derived species. For example, one can store metabolites that are easy to degrade, and another can accelerate certain reaction by colocalizing enzymes that mediate the same metabolic pathway.
Especially, Encapsulin that derived from Thermotoga maritima (TmEncap), form 20nm sphere capsule as 60mer [11]. Furthermore, TmEncap that is engineered for better function have interesting features. This can display targeted protein on the surface of the capsule, and can also load targeted protein inside of the capsule [12][13].
In this project, displaying targeted protein on the surface of Encapsulin plays a significant role. This ability relies on SpyCatcher / SpyTag: detailed below in Part.3
Fig.1 The structure of TmEncapsulin
PDB:3DKT
Part.3 SpyCatcher / SpyTag
SpyCatcher and SpyTag are derived from Streptococcus pyogenes. These proteins form isopeptide bonds between them when they are mixed together [14].
In previous research about TmEncap, it is showed that peptides inserted after 138th amino acid in TmEncap can be exposed outside of the protein capsule as a loop [15]. Furthermore, “Bae et.al” showed when SpyTag is inserted at the same position, a bond between SpyCatcher and "SpyTag inserted TmEncap" (SpyTmEnc) is also formed [12].
In order to display protein on the protein capsule, we can fuse protein to C-terminus of the TmEncapsulin directly. Since SpyTag/SpyCatcher can display several kinds of protein on the same capsule, we chose SpyTmEnc to give flexibility to our device.
Fig.2 Visual description of SpyTag/SpyCatcher bond formation
SpyTag is shown in red, and SpyCatcher is shown in blue. In the magnified view, the isopeptide bond is shown in purple.
PDB:4LMI
All proteins carry 6x-His tag for purification. For more detail about part design, see Part pages.
References
1 Boraston, A.B., Bolam, D.N., Gilbert, H.J., and Davies, G.J. (2004).
Carbohydrate-binding modules: Fine-tuning polysaccharide recognition.
Biochem. J. 382, 769–781.
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 Zhang, Y., Chen, S., Xu, M., Cavoco-Paulo, A.P., Wu, J., and Chen, J. (2010).
Characterization of thermobifida fusca cutinase-carbohydrate-binding module fusion proteins and their potential application in bioscouring.
Appl. Environ. Microbiol. 76, 6870–6876.
4 Osaki, T., Omotezako, M., Nagayama, R., Hirata, M., Iwanaga, S., Kasahara, J., Hattori, J., Ito, I., Sugiyama, H., and Kawabata, S.I. (1999).
Horseshoe crab hemocyte-derived antimicrobial polypeptides, tachystatins, with sequence similarity to spider neurotoxins.
J. Biol. Chem. 274, 26172–26178.
5 Islam, S., Apitius, L., Jakob, F., and Schwaneberg, U. (2019).
Targeting microplastic particles in the void of diluted suspensions.
Environ. Int. 123, 428–435.
6 Rübsam, K., Stomps, B., Böker, A., Jakob, F., and Schwaneberg, U. (2017).
Anchor peptides: A green and versatile method for polypropylene functionalization.
Polymer (Guildf). 116, 124–132.
7 Rübsam, K., Davari, M.D., Jakob, F., and Schwaneberg, U. (2018).
KnowVolution of the polymer-binding peptide LCI for improved polypropylene binding.
Polymers (Basel). 10, 1–12.
8 Al, L. H. and N. R. S.-W. et. (2019).
BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm.
Journal of Chemical Information and Modeling, 53(9), 1689–1699.
9 Yang, Y., Yang, J., and Jiang, L. (2016).
Comment on "a bacterium that degrades and assimilates poly(ethylene terephthalate) ".
Science (80-. ). 353, 759.
10 Austin, H.P., Allen, M.D., Donohoe, B.S., Rorrer, N.A., Kearns, F.L., Silveira, R.L., Pollard, B.C., Dominick, G., Duman, R., Omari, K. El, et al. (2018).
Characterization and engineering of a plastic-degrading aromatic polyesterase.
Proc. Natl. Acad. Sci. U. S. A. 115, E4350–E4357.
11 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.
12 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.
13 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.
14 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.
15 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.