Difference between revisions of "Team:HokkaidoU Japan/Multimerization"
| Line 24: | Line 24: | ||
<tr> | <tr> | ||
| − | <td> | + | <td>Difficult to connect several huge proteins</td> |
| − | <td> | + | <td>Possible to synthesize the proteins individually. Can also form a huge complex</td> |
</tr> | </tr> | ||
| Line 58: | Line 58: | ||
<div> | <div> | ||
| − | <p>There are also disadvantages using SAP. Since the number of the possible combination of several | + | <p>There are also disadvantages to using SAP. Since the number of the possible combination of several |
| − | different is infinite, there is no guarantee that we can always obtain the expected combination | + | different proteins is infinite, there is no guarantee that we can always obtain the expected combination |
when we form the protein complex. | when we form the protein complex. | ||
| − | + | One solution to the problem is limiting the number of combination by using different SAP. | |
As forming protein complex with different functions, this multimer forming method with SAP | As forming protein complex with different functions, this multimer forming method with SAP | ||
| − | let us create more functional | + | 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. | its function is expected to be enhanced. | ||
</p> | </p> | ||
| Line 89: | Line 89: | ||
formed the multimers of GFP. GFP’s molecular mass is 26891Da. When fusing with P<sub>11</sub>-4, it’s | formed the multimers of GFP. GFP’s molecular mass is 26891Da. When fusing with P<sub>11</sub>-4, it’s | ||
31709Da. With RADA16-I, it’s 31943Da. When they form multimer, the molecular mass will be more | 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 more than 50KDa. | + | 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, | For the evaluation, we ordered IDT the designed constructions and put them on the vectors. Then, | ||
we introduced them to <span style="font-style: italic">E.Coli</span>. Using IPTG induction , the proteins were expressed. Causing | we introduced them to <span style="font-style: italic">E.Coli</span>. Using IPTG induction , the proteins were expressed. Causing | ||
bacteriolysis with freeze-thaw, we acquired the supernatant contains the proteins by centrifugal | 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 | separation. Purifying the protein with Ni-affinity chromatography, we filtrated the solution | ||
| − | to separate the proteins with less than 50KDa. We irradiated 480nm light to filtrate and observed | + | 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.<br> | whether 580nm wave-length light was emitted.<br> | ||
</p> | </p> | ||
Revision as of 09:25, 9 October 2016
"
Team:HokkaidoU Japan
We advocate the new method to create multimer using the self-assembling ability of SAP. By fusing SAP to the end of a protein, it will condense with other proteins’ SAP domains and form the 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. The new method that we suggest which uses SAP is superior to the ordinary one for these resons.
| Linker Method | SAP Method |
|---|---|
| Difficult to connect several huge proteins | Possible to synthesize the proteins individually. Can also form a huge complex |
| Regulated by one promoter | Each protein can be expressed individually |
| The possibility of deformation of the 3D-structure | Low possibility of deformation since they only connect with proteins which can condense |
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. 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.
ここに本文
ここに本文
