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

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<td style="border-style: none"; align="center"><span class="small">Fig. 10. The construction of multimerization using SAP<br>This is the construct for making multiple complex. We used RADA16-I and P<span class="sitatuki">11</span>-4 as SAP. <br>C is a cysteine residues in short linker.</span></td>
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<td style="border-style: none"; align="center"><span class="small">Fig. 10. The construction of multimerization using SAP<br>This is the construct for making multiple complex. We used RADA16-I and P<span class="sitatuki">11</span>-4 as SAP. C is a cysteine residues in short linker.</span></td>
 
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Revision as of 14:19, 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 an 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 their 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 also had 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 year. We tried multimerization using it and not only used it but also made firm connections.


methods


We tried forming multimers using the self-assembling peptide (SAP), P11-4 (QQRFEWEFEQQ) and RADA16-I (RADARADARADARADA). And to make firm 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 (Fig. 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 31709 Da. With RADA16-I, it’s 31943 Da. When they form multimer, the molecular mass will be more than 60 kDa. Consequently, we used the filter which filters out the proteins with mass of more than 50 KDa.
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 480 nm light to filtrate and observed whether 580 nm wave-length light was emitted.



results


We put above CDS (Fig. 8) into pET15b and expressed (Fig. 10). As negative control we made a construction containing GFP without SAPs and SLs (Fig. 11). GFPs with SAPs and SLs was expected to become multiple complexes (Fig. 12).
construct
Fig. 10. The construction 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. 11. The construction of a negative control
We made a negative control which had only GFP to test the effect of SAPs.

image
Fig. 12. Expected forming multiple complex



conclusion

As future work, anyone can make multi-enzyme-complex if the protein is designed to have BamHI restriction enzyme sites in both ends. Our construction have also BamHI site at GEP ends. So, you can cut out the GFP and put on any protein using cloning site (Fig. 13).
Tool
Fig. 13. The construction for making subunits of artificial multi-enzyme-complex
We designed this construction to have a cloning site. If you design the protein which ends are BamHI site, you can make the multimer easily.



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.