Difference between revisions of "Team:HokkaidoU Japan/Multimerization"
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<br>[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. | <br>[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. | ||
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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.
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).
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.
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.
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).
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.
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 complexs (Fig. 12).
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).
[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. The 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 |
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).
| 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 |
|
|
Fig. 3. Using linkers |
Fig. 4. Using SAPs |
 |
Fig. 5. Demerit of using linkers |
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.
 |
|
Fig. 6. Demerit of using SAP method |
Fig. 7. Resolution for infinite combinations |
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.
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).
 |
Fig. 8. Design of the coding sequence |
Assay
 |
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.
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 complexs (Fig. 12).
 |
Fig. 10. The construction of multimerization using SAP |
 |
Fig. 11. The construction of a negative control |
 |
Fig. 12. Expected forming multiple complex |
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).
 |
Fig. 13. The construction for making subunits of artificial multi-enzyme-complex |
[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.
