Difference between revisions of "Team:HokkaidoU Japan/Multimerization"

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Revision as of 06:24, 19 October 2016

Team:HokkaidoU Japan - 2016.igem.org

 

Team:HokkaidoU Japan

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overview
enzymatic reaction

Fig. 1. The enzyme reaction by multiple complex
To connect different enzymes will
make continuous reaction efficiently.


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.

large block

Fig. 2. Huge complex using SAP
To connect same enzymes like fluorescent proteins will amplify thir effects.




However, the ordinary method uses linkers to connect proteins. We think the new method using SAP is superior to the ordinary one for these reasons (Table. 1).


Table. 1. Comparison between linkers and SAPs

Linker Method SAP Method
Regulated by one promoter (Fig. 3) Each protein can be produced 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

linker methods SAP methods

Fig. 3. Using linkers
Expressions of gene A, B and C which code protein A, B and C are regulated by one promoter. If you connect some huge proteins, the expression efficiency may be decreased because the coding sequence is too long.

Fig. 4. Using SAPs
You can produce protein A, B and C individually. After expression, they gather by SAPs and form disufide bonds by SLs.



steric hindrance

Fig. 5. Demerit of using linkers
In linker method, you need to consider the linker length to avoid the steric hindrance.



We thought the SAP method was best one but it had also disadvantages. 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.
One solution to the problem is limiting the number of combination by using different SAP. That can reduce probability of incorrect connection a little.
demerit resolution

Fig. 6. Demerit of using SAP method
If some kinds of protein are expressed,
there are so many combination.
You may not be able to get the correct combination.

Fig. 7. Resolution for infinite combinations
When you use some kinds of SAP,
incorrect connections will decrease.



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

We tried to establish novel uses of SAP in this yaer. We challenged multimerization using it and not only used it but also made firmly connection.

methods


We tried forming multimers using the self-assembling peptide (SAP), P11-4 (QQRFEWEFEQQ) and RADA16-I (RADARADARADARADA). And to make firmly bonds we designed short linker (GGCGG) called SL for short. We Connected SL and SAP to both ends of the protein. In this experiment, we used GFP as test (Fif. 8).
construct

Fig. 8. Design of the coding sequence


Assay

methods

Fig. 9. Method for verifying whether proteins form multiple complex


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.


image

Fig. 9. キャプション




results


ここに本文
construct

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.


Negative control

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


Tool

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.




conclusion

ここに本文


reference

[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.