Difference between revisions of "Team:HokkaidoU Japan/Multimerization"
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| + | <td style="border-style: none;"><h2>Fig. 3. Using linkers</h2></td> | ||
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Revision as of 14:49, 18 October 2016
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Team:HokkaidoU Japan
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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.
The ordinary method uses linkers to connect proteins. We think the new method using SAP is superior to the ordinary one for these reasons.
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.
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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[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.
 |
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 |
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 |
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 |
Fig. 9. Construct of multimerization using SAP |
 |
Fig. 10. Construct of a negative control |
 |
Fig. 11. Construct for making subunits of artificial multi-enzyme-complex |
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.
