Biomod/2011/Slovenia/BioNanoWizards/resultssolublezfp

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</ul> <!-- End PureCSSMenu.com MENU --> </div> </div> </div> <!-- end #header --> <div id="page" class="container"> <div id="content"> <div class="post"> <div class="entry"><big><big><big><big><span 

style="color: black; font-weight: bold;">Soluble ZFP</span></big></big></big></big><br>

<br> <table 

style="margin: 3px 0px 20px 20px; width: 444px; height: 50px; float: right;"
border="0" cellpadding="0" cellspacing="0">
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     <td style="text-align: center;"><img
style="padding: 0pt 0pt 5px; width: 540px; height: 388px;"
alt="" src="http://openwetware.org/images/6/6b/MBPZFP.png"></td>
   </tr>
   <tr style="font-family: Arial; font-weight: bold;">
     <td style="text-align: justify;">Figure X0:

Threedimensional representation of chimeric ZFP. <span 

style="font-weight: normal;">MBP is shown in green and ZFP

(Zif268) is shown in cyan bound to the target DNA.</span></td> 

   </tr>
 </tbody>

</table> <span style="font-family: Arial;"> A need for properly folded and soluble zinc finger proteins was central for the success of our project. Our colleagues at iGEM2010 competition used zinc fingers within bacterial cells and did not face the problem of their solubility and propensity to aggregate in vitro. Limited with the time frame of the competition we decided to search for the universal solution other than trying to optimize the solution conditions for each ZFP separately.</span><br 

style="font-family: Arial;">

<br style="font-family: Arial;"> <span style="font-family: Arial;"> It has been known from the literature that fusion of proteins that have tendency to aggregate with highly soluble protein often results in rescuing the "misbehaving proteins". Therefore we fused zinc finger proteins with highly soluble protein domains, namely GST (glutathione-S-transferase) and MBP (maltose binding protein). Preparation of cloning constructs (which was not staightforward and without problems) is described in more detail in Plasmid construction section in Methods. </span><br style="font-family: Arial;"> <br style="font-family: Arial;"> <span style="font-family: Arial;"> During BIOMOD2011 project we successfully achieved bacterial expression of 14 chimeric zinc finger proteins and isolated 11 of them, all of which had solubility tags. Use of solubility tags allowed us to isolate chimeric zinc finger proteins under the non-denaturing conditions from bacterial lysates, which preserved the biological function of isolated proteins (described in the section on Functional ZFPs). </span><br 

style="font-family: Arial;">

<br style="font-family: Arial;"> <span style="font-family: Arial;"> We isolated six zinc finger protein chimeras tagged with GST. Three of them, GST-Zif268, GST-sZif268 and GST-PBSII, contained short three-finger domains recognizing 9 base pairs of their DNA targets. On Figure X1 we present the corresponding images of Coomassie stained SDS-PAGE gels and Western blot analysis results (below) depicting analyzed samples of soluble and insoluble fractions of bacterial lysates containing chimeric ZFPs (detailed methods are described in Protein expression of the Methods section). <br> <br> </span><span style="font-family: Arial;">Two of the proteins, GST-Zif268 and GST-sZif268, were also successfully isolated by chelating chromatography as shown on the images of Coomassie stained SDS-PAGE and Western blot analysis results in Figure X2.</span><br> <table style="text-align: left; width: 100%;" border="0" 

cellpadding="8" cellspacing="0">
 <tbody>
   <tr>
     <td
style="text-align: center; vertical-align: bottom; width: 50%;"><img
style="margin-top: 20px; width: 443px; height: 559px;" alt=""
src="http://openwetware.org/images/a/ab/Produkcijakratkiprsti.png"></td>
     <td
style="text-align: center; vertical-align: bottom; width: 50%;"><img
style="margin-top: 20px; width: 245px; height: 559px;" alt=""
src="http://openwetware.org/images/0/01/Izolacijakratkiprsti.png"></td>
   </tr>
   <tr>
     <td style="text-align: justify;"><span
style="font-family: Arial;"><span
style="font-weight: bold;">

Figure X1: Production of GST-fused three-finger domain-containing ZFPs.</span> Each image comprises samples from the soluble fraction of bacterial lysate (SN or supernatant) and insoluble fraction (IBs or inclusion bodies). Left to right: GST-Zif268 (Mw of 37.8 kDa), GST-sZif (Mw of 38.4 kDa), GST-PBSII (Mw of 38.0 kDa). Top: Coomassie stained SDS-PAGE gels. Bottom: Western blot analysis using anti-His4 antibodies. 

     </span></td>
     <td style="text-align: justify; vertical-align: top;"><span
style="font-family: Arial;"><span
style="font-weight: bold;">

Figure X2: Isolation of GST-fused three-finger domain containing ZFPs.</span> Each image shows a sample of isolated proteins GST-Zif268 (Mw of 37.8 kDa) and GST-sZif (Mw of 38.4 kDa). Top: Coomassie stained SDS-PAGE gels. Bottom: western blot analysis using anti-His6 antibodies. 

     </span></td>
   </tr>
 </tbody>

</table> <br> <span style="font-family: Arial;"> Additionally, we isolated 3 six-finger chimeras tagged with GST, GST-2C7, GST-AZPA4 and GST-6F6. Each of these ZFPs contains six-fingers, thus recognizing 18 base pair target DNA. Production of six-finger chimeras was confirmed by SDS-PAGE and Western blot analysis, which can be seen in Figure X3. All of them were successfully isolated as shown in Figure X4. </span><br style="font-family: Arial;"> <table style="text-align: left; width: 100%;" border="0" 

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 <tbody>
   <tr>
     <td style="text-align: center; vertical-align: bottom;"><img
style="margin-top: 20px; width: 460px; height: 562px;" alt=""
src="http://openwetware.org/images/6/6f/Produkcijadolgiprsti.png"></td>
     <td style="text-align: center; vertical-align: bottom;"><img
style="margin-top: 20px; width: 386px; height: 562px;" alt=""
src="http://openwetware.org/images/3/30/Izolacijadolgiprsti.png"></td>
   </tr>
   <tr>
     <td style="text-align: justify;"><span
style="font-family: Arial;"><span
style="font-weight: bold;">

Figure X3: Production of GST-fused six-finger domains containing ZFPs. </span> Each image comprises samples from bacterial lysate soluble fraction (SN or supernatant) and insoluble fraction (IBs or inclusion bodies). Left to right: GST-AZPA4 (Mw of approximately 46.8 kDa), GST-2C7 (Mw of approximately 48.9 kDa), GST-6F6 (Mw of approximately 48.3 kDa). Top: Coomassie stained SDS-PAGE gels. Bottom: Western blot analysis using anti-His4 antibodies. </span></td> 

     <td style="text-align: justify; vertical-align: top;"><span
style="font-family: Arial;"><span
style="font-weight: bold;">

Figure X4: Isolation of GST-fused six-finger domains containing ZFPs. </span> Each image shows a sample of isolated proteins GST-AZPA4 (Mw of approximately 46.8 kDa), GST-2C7 (Mw of approximately 48.9 kDa) and GST-6F6 (Mw of approximately 48.3 kDa). Top: Coomassie stained SDS-PAGE gels. Bottom: Western blot analysis using anti-His4 antibodies. </span></td> 

   </tr>
 </tbody>

</table> <br> <span style="font-family: Arial;"> 6F6 and 2C7 were also fused with MBP. Production and isolation of MBP-6F6 and MBP-2C7 was analyzed by SDS-PAGE and Western blot analysis, which can be seen in Figure X5. </span><br style="font-family: Arial;"> <table style="text-align: left; width: 100%;" border="0" 

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     <td><img
style="margin-top: 10px; width: 761px; height: 332px;" alt=""
src="http://openwetware.org/images/e/ec/MBPsolublezadnji.png"></td>
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     <td valign="undefined"><span
style="font-family: Arial;"><span
style="font-weight: bold;">

Figure X5: Production and isolation of MBP-fused ZFPs containing six-finger domains.</span> Left to right: Coomassie stained SDS-PAGE gels of MBP-6F6 (Mw of approximately 64.3 kDa) and MBP-2C7 (Mw of approximately 64.9 kDa) soluble and insoluble fractions; Western blot analysis of MBP-6F6 and MBP-2C7 soluble and insoluble fractions using anti-His4 antibodies; Coomassie stained SDS-PAGE of isolated MBP-6F6 (Mw of approximately 64.3 kDa); Coomassie stained SDS-PAGE of isolated MBP-6F6 (Mw of approximately 64.9 kDa). 

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</table> <br style="font-family: Arial;"> <span style="font-family: Arial;"> After initial failed attempts to produce soluble zinc finger protein chimeras with mCitrine and Renilla luciferase proteins, we decided to add MBP to their N-termini. We confirmed that the addition of MBP rendered such chimeric proteins more soluble and readily expressed in the functional and soluble form. We isolated soluble MBP-YFP-AZPA4 and MBP-Rluc-2C7 chimeras for BRET experiments. </span><br style="font-family: Arial;"> <br style="font-family: Arial;"> <span style="font-family: Arial;"> We used the same reasoning for the production of twin zinc finger protein chimeras, that is 2C7-MBP-6F6 and AZPA4-MBP-6F6 chimeras for the vertical DNA origami stacking using protein tethers. In this case, solubility was also increased, nevertheless it was significantly lower, which might be due to the fact that having two zinc finger domains could not be sufficiently compensated by the addition of one solubility domain.</span><!-- TU SE KONEA GLAVNO BESEDILO NA STRANI --> </div> </div> </div> <!-- end #content --> <div style="clear: both;">&nbsp;</div> </div> <!-- end #page --></div> <div id="footer-content" class="container"> <div id="footer-bg"> <table 

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