<td style="border-style: none;"><img src="https://static.igem.org/mediawiki/2016/3/3b/T--HokkaidoU_Japan--multimerization_image6.png" alt="enzymatic reaction" height="300px" width="auto" style="float:left"></td>  

<td style="border-style: none"; align="center"><h2>Fig. 1. The enzyme reaction by multiple complex<br>To connect different enzymes will<br>make continuous reaction efficiently. </h></td>

<td style="border-style: none"; align="center"><h2>Fig. 2. Huge complex using SAP<br>To connect same enzymes like fluorescent proteins will amplify thir effects.</h2></td>

<br>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).</br>

     <td>Each protein can be produced individually (Fig. 4)</td>

<td style="border-style: none;"><h2>Fig. 3. Using linkers<br>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. </h2></td>

<td style="border-style: none;"><h2>Fig. 4. Using SAPs<br>You can produce protein A, B and C individually. After expression, they gather by SAPs and form disufide bonds by SLs.</h2></td>

<td style="border-style: none"; align="center"><h2>Fig. 5. Demerit of using linkers<br>In linker method, you need to consider the linker length to avoid the steric hindrance.</h2></td>

<br>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.

<br>One solution to the problem is limiting the number of combination by using different SAP. That can reduce probability of incorrect connection a little.</br>

<td style="border-style: none;"><h2>Fig. 6. Demerit of using SAP method<br>If some kinds of protein are expressed,<br>there are so many combination.<br>You may not be able to get the correct combination.</h2></td>

<td style="border-style: none;"><h2>Fig. 7. Resolution for infinite combinations<br>When you use some kinds of SAP,<br>incorrect connections will decrease.</h2></td>

<br>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.

<br>

 

<br>We tried forming multimers using the self-assembling peptide (SAP), P<span class="sitatuki">11</span>-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).  

 

<td style="border-style:none; float:center"><img src="https://static.igem.org/mediawiki/2016/d/df/T--HokkaidoU_Japan--multimerization_construct.png

" alt="construct" height="auto" width="900px"></td>  

<td style="border-style: none"; align="center"><h2>Fig. 8. Design of the coding sequence</h2></td>

 

<br>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).

<td style="border-style: none"; align="center"><h2>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.</h2></td>

<td style="border-style: none"; align="center"><h2>Fig. 11. The construction of a negative control<br>We made a negative control which had only GFP to test the effect of SAPs.</h2></td>

<td style="border-style:none; float:center"><img src="https://static.igem.org/mediawiki/2016/2/28/T--HokkaidoU_Japan--image21.png" alt="image" height="500px" width="auto"></td>  

<td style="border-style: none"; align="center"><h2>Fig. 12. Expected forming multiple complex</h2></td>

<br>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).

<br>[1] Lee H, DeLoache WC, Dueber JE. Spatial organization of enzymes for metabolic engineering. Metab Eng. 2012;14:242?251.