Team:HokkaidoU Japan/Multimerization

"

Team:HokkaidoU Japan

\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\

• Top
• Project
  • Overview
    • Project Description
    • Background
    • Proof of concept
    • Modeling
    • Achievements
  • MULTIMERIZATION
    • Overview
    • Methods
    • Results
    • Conclusion
  • FUTURE WORK
    • Circularization
    • Kill Switch
    • Transport
    • Aggregation
  • Extra Materials
    • Parts
    • Notebook
    • Protocols
    • Safety
• Human practices
• Collaborations
• Team
  • Attribution
  • Member

Fig. 1. Enzyme reaction by multiple complex

We made a platform of technology for constructing covalently linked multi-enzyme-complex through disulfide bonds recruited by self-assembling peptide (SAP). By fusing SAP to the end of a protein, it will condense with other proteins’ SAP domains and form the complex. The SAP domains is pinched by short linkers (SL) that have cysteine residues. When the SAPs gather and SLs get close, disulfide bonds are formed between other SLs. So, we will make unbreakable complex. By using this method, we’ll be able to connect several enzymes and allow huge complexed proteins to be formed. It’ll improve the efficiency of a continuous reaction.

Fig. 2. Huge complex using SAP We thought the intensity of fluorescent proteins, like GFP increased.

The ordinary method uses linkers to connect proteins. We think the new method using SAP is superior to the ordinary one for these reasons.

Linker Method	SAP Method
Regulated by one promoter (Fig. 3)	Each protein can be expressed individually (Fig. 4)
Difficult to produce several huge complex	Possible to synthesize the proteins individually. Can also form a huge complex (Fig. 4)
The possibility of deformation of the 3D-structure (Fig. 5)	Low possibility of deformation since they only connect with proteins which can condense

Fig. 3. Using linkers	Fig. 4. Using SAPs

Fig. 5. Demerit of using linkers

There are also disadvantages to using SAP. Since the number of the possible combination of several different proteins is infinite, there is no guarantee that we can always obtain the expected combination when we form the protein complex. One solution to the problem is limiting the number of combination by using different SAP. That can reduce probability of miss connection a little.

Fig. 6. Demerit of using SAP method	Fig. 7. Resolution for miss connections

As forming protein complex with different functions, this multimer forming method with SAP let us create more functional units. When same kinds of proteins are used, it’ll be a large block and its function is expected to be enhanced.

Fig. 8. Method for verifying whether proteins form multiple complex

We tried forming multimers using the self-assembling peptide, P11-4 and RADA16-I. We Connected short linker(GGCGG) and SAP to both ends of the protein. In this experiment, we formed the multimers of GFP. GFP’s molecular mass is 26891Da. When fusing with P11-4, it’s 31709Da. With RADA16-I, it’s 31943Da. When they form multimer, the molecular mass will be more than 60kDa. Consequently, we used the filter which filters out the proteins with mass of more than 50KDa. For the evaluation, we ordered IDT the designed constructions and put them on the vectors. Then, we introduced them to E.coli. Using IPTG induction , the proteins were expressed. Causing bacteriolysis with freeze-thaw, we acquired the supernatant contains the proteins by centrifugal separation. Purifying the protein with Ni-affinity chromatography, we filtrated the solution to separate the proteins with mass of less than 50KDa. We irradiated 480nm light to filtrate and observed whether 580nm wave-length light was emitted.

Fig. 9. キャプション

ここに本文

Fig. 9. Construct of multimerization using SAP This is the construct for making multiple complex. We used RADA16-I and P11-4 as SAP. C is a cysteine residues in short linker.

Fig. 10. Construct of a negative control We made a negative control which had only GFP to test the effect of SAPs.

Fig. 11. Construct for making subunits of artificial multi-enzyme-complex We designed this construct to had a cloning site. If you design the protein which ends are BamHI site, you can make the multimer easily.

ここに本文

[1] Lee H, DeLoache WC, Dueber JE. Spatial organization of enzymes for metabolic engineering. Metab Eng. 2012;14:242?251.
[2] Castellana M1, Wilson MZ2, Xu Y3, Joshi P2, Cristea IM2, Rabinowitz JD4, Gitai Z2, Wingreen NS3. Enzyme clustering accelerates processing of intermediates through metabolic channeling. Nat Biotechnol. 2014 Oct;32(10):1011-8.