Team:HokkaidoU Japan/Multimerization

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Team:HokkaidoU Japan

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• 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

We advocate the new method to create multimer using the self-assembling ability of SAP. By fusing SAP to the end of a protein, it will condense with other proteins’ SAP domains and form the 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. The ordinary method uses linkers to connect proteins. The new method that we suggest which uses SAP is superior to the ordinary one for these resons.

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

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. 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.

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.

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