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Revision as of 12:55, 19 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 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.
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
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.
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.
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).
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.
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).
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 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.
 |
| 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).
| 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 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. |
 |
| 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.
 |
|
| 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.
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).
 |
| 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 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.
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).
 |
| 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. |
 |
| Fig. 11. The construction of a negative control We made a negative control which had only GFP to test the effect of SAPs. |
 |
| 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 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. |
[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.
