Biomod/2011/Slovenia/BioNanoWizards/resultssolublezfp: Difference between revisions

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<div class="post">
<div class="post">
<div class="entry"><big><big><big><big><span
<div class="entry"><big><big><big><big><span
  style="color: black; font-weight: bold;">Soluble ZFP</span></big></big></big></big><br>
  style="color: black; font-weight: bold;">Soluble ZFPs</span></big></big></big></big><br>
<br>
<br><br>
<table
<table
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     </tr>
     </tr>
     <tr style="font-family: Arial; font-weight: bold;">
     <tr style="font-family: Arial; font-weight: bold;">
       <td style="text-align: justify;">Figure X0:
       <td style="text-align: justify;">Figure 7:
Threedimensional representation of chimeric ZFP. <span
Three dimensional representation of chimeric ZFP. <span
  style="font-weight: normal;">MBP is shown in green and ZFP
  style="font-weight: normal;">MBP is shown in green and ZFP
(Zif268) is shown in cyan bound to the target DNA.</span></td>
(Zif268) is shown in cyan, bound to the target DNA (orange and blue).</span></td>
     </tr>
     </tr>
   </tbody>
   </tbody>
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<span style="font-family: Arial;">
<span style="font-family: Arial;">
A need for properly folded and soluble zinc finger proteins was central
A need for properly folded and soluble zinc finger proteins was central
for the success of our project. Our colleagues at iGEM2010 competition
for the success of our project. Our colleagues at <a href="http://2010.igem.org/Team:Slovenia">iGEM2010 competition</a>
used zinc fingers within bacterial cells and did not face the problem
used zinc fingers for positioning proteins on the linear DNA within bacterial cells and did not face the problem
of their solubility and propensity to aggregate in vitro. Limited with
of solubility and propensity of ZFPs to aggregate <em>in vitro</em>. Limited with
the time frame of the competition we decided to search for the
the time frame of the competition we decided to search for the
universal solution other than trying to optimize the solution
universal solution other than trying to optimize the solution
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<br style="font-family: Arial;">
<br style="font-family: Arial;">
<span style="font-family: Arial;">
<span style="font-family: Arial;">
It has been known from the literature that fusion of proteins that have
It has been known from the literature that fusion of proteins that have a
tendency to aggregate with highly soluble protein often results in
tendency to aggregate with highly soluble protein often results in
rescuing the "misbehaving proteins". Therefore we fused zinc finger
rescuing the "misbehaved proteins". Therefore we fused zinc finger
proteins with highly soluble protein domains, namely GST
proteins with highly soluble protein domains, namely GST
(glutathione-S-transferase) and MBP (maltose binding protein).
(glutathione-S-transferase) and MBP (maltose binding protein).
Preparation of cloning constructs (which was not staightforward and
Preparation of cloning constructs (which was however not straightforward and
without problems) is described in more detail in Plasmid construction
without problems) is described in more detail in <a href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/methplasmidconst">Plasmid construction</a>
section in Methods.
section in Methods.
</span><br style="font-family: Arial;">
</span><br style="font-family: Arial;">
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During BIOMOD2011 project we successfully achieved bacterial expression
During BIOMOD2011 project we successfully achieved bacterial expression
of 14 chimeric zinc finger proteins and isolated 11 of them, all of
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
which had added solubility domains or tags. Use of solubility tags allowed us to isolate
chimeric zinc finger proteins under the non-denaturing conditions from
chimeric zinc finger proteins under the non-denaturing conditions from
bacterial lysates, which preserved the biological function of isolated
bacterial lysates, which preserved the biological function of isolated
proteins (described in the section on Functional ZFPs). </span><br
proteins (described in the section on <a href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultsfunctionalizedzfp">Functionalized ZFPs</a>). </span><br
  style="font-family: Arial;">
  style="font-family: Arial;">
<br style="font-family: Arial;">
<br style="font-family: Arial;">
<span style="font-family: Arial;">
<span style="font-family: Arial;">
We isolated six zinc finger protein chimeras tagged with GST. Three of
We produced six zinc finger protein chimeras tagged with GST. Three of
them, GST-Zif268, GST-sZif268 and GST-PBSII, contained short
them, GST-Zif268, GST-sZif268 and GST-PBSII, contained three-finger domains recognizing 9 base pairs of their DNA targets. On
three-finger domains recognizing 9 base pairs of their DNA targets. On
<strong>Figure 8</strong> we present the analysis of soluble and insoluble fractions of bacterial
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
lysates containing chimeric ZFPs (detailed methods are described in
Protein expression of the Methods section).
<a href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/methprotprodandisol">Protein expression</a> of the Methods section).
<br>
<br>
<br>
<br>
</span><span style="font-family: Arial;">Two of the
</span><span style="font-family: Arial;">Two of the
proteins, GST-Zif268 and GST-sZif268, were also successfully isolated
proteins, GST-Zif268 and GST-sZif268, were also successfully purified
by chelating chromatography as shown on the images of Coomassie stained
by chelating chromatography as shown in <strong>Figure 9</strong> of SDS-PAGE and Western blot analysis.</span><br>
SDS-PAGE and Western blot analysis results in Figure X2.</span><br>
<table style="text-align: left; width: 100%;" border="0"
<table style="text-align: left; width: 100%;" border="0"
  cellpadding="8" cellspacing="0">
  cellpadding="8" cellspacing="0">
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  style="font-family: Arial;"><span
  style="font-family: Arial;"><span
  style="font-weight: bold;">
  style="font-weight: bold;">
Figure X1: Production of GST-fused three-finger domain-containing ZFPs.</span>
Figure 8: Production of GST-fused three-finger domain-containing ZFPs.</span>
Each image comprises samples from the soluble fraction of bacterial
Each image comprises samples from the soluble fraction of bacterial
lysate (SN or supernatant) and insoluble fraction (IBs or inclusion
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
bodies). Left to right: GST-Zif268 (Mr of 38 kDa), GST-sZif (Mr of
38.4 kDa), GST-PBSII (Mw of 38.0 kDa). Top: Coomassie stained SDS-PAGE
38 kDa), GST-PBSII (Mr of 38 kDa). Top: Coomassie stained SDS-PAGE
gels. Bottom: Western blot analysis using anti-His4 antibodies.
gels. Bottom: Western blot analysis using anti-His<sub>4</sub> antibodies.
       </span></td>
       </span></td>
       <td style="text-align: justify; vertical-align: top;"><span
       <td style="text-align: justify; vertical-align: top;"><span
  style="font-family: Arial;"><span
  style="font-family: Arial;"><span
  style="font-weight: bold;">
  style="font-weight: bold;">
Figure X2: Isolation of GST-fused three-finger domain containing ZFPs.</span>
Figure 9: Purification of GST-fused three-finger domain containing ZFPs.</span>
Each image shows a sample of isolated proteins GST-Zif268 (Mw of 37.8
Each image shows a sample of isolated proteins GST-Zif268 (Mr of 38
kDa) and GST-sZif (Mw of 38.4 kDa). Top: Coomassie stained SDS-PAGE
kDa) and GST-sZif (Mr of 38 kDa). Top: Coomassie stained SDS-PAGE
gels. Bottom: western blot analysis using anti-His6 antibodies.
gels. Bottom: western blot analysis using anti-His<sub>4</sub> antibodies.
       </span></td>
       </span></td>
     </tr>
     </tr>
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<br>
<br>
<span style="font-family: Arial;">
<span style="font-family: Arial;">
Additionally, we isolated 3 six-finger chimeras tagged with GST,
Additionally, we isolated three chimeric proteins tagged with GST:
GST-2C7, GST-AZPA4 and GST-6F6. Each of these ZFPs contains
GST-2C7, GST-AZPA4 and GST-6F6. Each of these ZFPs contains
six-fingers, thus recognizing 18 base pair target DNA. Production of
six-fingers, thus recognizing 18 base pair target DNA. Production of
six-finger chimeras was confirmed by SDS-PAGE and Western blot
six-finger chimeras was confirmed by SDS-PAGE and Western blot
analysis, which can be seen in Figure X3.
analysis, which can be seen in Figure 10.
All of them were successfully isolated as shown in Figure X4.
All of them were successfully purified as shown in Figure 11.
</span><br style="font-family: Arial;">
</span><br style="font-family: Arial;">
<table style="text-align: left; width: 100%;" border="0"
<table style="text-align: left; width: 100%;" border="0"
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  style="font-family: Arial;"><span
  style="font-family: Arial;"><span
  style="font-weight: bold;">
  style="font-weight: bold;">
Figure X3: Production of GST-fused six-finger domains containing ZFPs. </span>
Figure 10: Bacterial production of GST-fused six-finger domains containing ZFPs. </span>
Each image comprises samples from bacterial lysate soluble fraction (SN
Each image comprises samples from bacterial lysate soluble fraction (SN
or supernatant) and insoluble fraction (IBs or inclusion bodies). Left
or supernatant) and insoluble fraction (IBs or inclusion bodies). Left
to right: GST-AZPA4 (Mw of approximately 46.8 kDa), GST-2C7 (Mw of
to right: GST-AZPA4 (Mr of approximately 47 kDa), GST-2C7 (Mr of
approximately 48.9 kDa), GST-6F6 (Mw of approximately 48.3 kDa). Top:
approximately 49 kDa), GST-6F6 (Mr of approximately 48 kDa). Top:
Coomassie stained SDS-PAGE gels. Bottom: Western blot analysis using
Coomassie stained SDS-PAGE gels. Bottom: Western blot analysis using
anti-His4 antibodies. </span></td>
anti-His<sub>4</sub> antibodies. </span></td>
       <td style="text-align: justify; vertical-align: top;"><span
       <td style="text-align: justify; vertical-align: top;"><span
  style="font-family: Arial;"><span
  style="font-family: Arial;"><span
  style="font-weight: bold;">
  style="font-weight: bold;">
Figure X4: Isolation of GST-fused six-finger domains containing ZFPs. </span>
Figure 11: Purification of GST-fused six-finger domains containing ZFPs. </span>
Each image shows a sample of isolated proteins GST-AZPA4 (Mw of
Each image shows a sample of isolated proteins GST-AZPA4 (Mr of
approximately 46.8 kDa), GST-2C7 (Mw of approximately 48.9 kDa) and
approximately 47 kDa), GST-2C7 (Mr of approximately 49 kDa) and
GST-6F6 (Mw of approximately 48.3 kDa). Top: Coomassie stained SDS-PAGE
GST-6F6 (Mr of approximately 48 kDa). Top: Coomassie stained SDS-PAGE
gels. Bottom: Western blot analysis using anti-His4 antibodies. </span></td>
gels. Bottom: Western blot analysis using anti-His<sub>4</sub> antibodies. </span></td>
     </tr>
     </tr>
   </tbody>
   </tbody>
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<span style="font-family: Arial;">
<span style="font-family: Arial;">
6F6 and 2C7 were also fused with MBP. Production and isolation of
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,
MBP-6F6 and MBP-2C7 was analyzed by SDS-PAGE and Western blot,
which can be seen in Figure X5.
which can be seen in Figure <strong>12</strong>.
</span><br style="font-family: Arial;">
</span><br style="font-family: Arial;">
<table style="text-align: left; width: 100%;" border="0"
<table style="text-align: left; width: 100%;" border="0"
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     <tr align="center">
     <tr align="center">
       <td><img
       <td><img
  style="margin-top: 10px; width: 761px; height: 332px;" alt=""
  style="margin-top: 10px; width: 609px; height: 265px;" alt=""
  src="http://openwetware.org/images/e/ec/MBPsolublezadnji.png"></td>
  src="http://openwetware.org/images/e/ec/MBPsolublezadnji.png"></td>
     </tr>
     </tr>
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  style="font-family: Arial;"><span
  style="font-family: Arial;"><span
  style="font-weight: bold;">
  style="font-weight: bold;">
Figure X5: Production and isolation of MBP-fused ZFPs containing
Figure 12: Production and isolation of MBP-fused ZFPs containing
six-finger domains.</span> Left to right: Coomassie stained
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
SDS-PAGE gels of soluble and insoluble fractions of lysates of bacteria producing MBP-6F6 (Mr of approximately 64 kDa) and MBP-2C7 (Mr
of approximately 64.9 kDa) soluble and insoluble fractions; Western
of approximately 65 kDa); Western
blot analysis of MBP-6F6 and MBP-2C7 soluble and insoluble fractions
blot analysis of MBP-6F6 and MBP-2C7 soluble and insoluble fractions
using anti-His4 antibodies; Coomassie stained SDS-PAGE of isolated
using anti-His<sub>4</sub> antibodies; Coomassie stained SDS-PAGE of isolated
MBP-6F6 (Mw of approximately 64.3 kDa); Coomassie stained SDS-PAGE of
MBP-6F6 (Mr of approximately 64 kDa); Coomassie stained SDS-PAGE of
isolated MBP-6F6 (Mw of approximately 64.9 kDa).
isolated MBP-6F6 (Mr of approximately 65 kDa).
       </span></td>
       </span></td>
     </tr>
     </tr>
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add MBP to their N-termini. We confirmed that the addition of MBP
add MBP to their N-termini. We confirmed that the addition of MBP
rendered such chimeric proteins more soluble and readily expressed in
rendered such chimeric proteins more soluble and readily expressed in
the functional and soluble form. We isolated soluble MBP-YFP-AZPA4 and
the functional and soluble form.  
MBP-Rluc-2C7 chimeras for BRET experiments.
</span><br style="font-family: Arial;">
</span><br style="font-family: Arial;">
<br style="font-family: Arial;">
<br style="font-family: Arial;">
<span style="font-family: Arial;">
<span style="font-family: Arial;">
We used the same reasoning for the production of twin zinc finger
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
protein chimeras, that is 2C7-MBP-6F6 and AZPA4-MBP-6F6 chimeric proteins which were designed for
the vertical DNA origami stacking using protein tethers. In this case,
the vertical DNA origami stacking using protein tethers. In this case,
solubility was also increased, nevertheless it was significantly lower,
solubility was also increased by the addition of MBP domain, nevertheless it was somewhat lower than that for other proteins described in this section,
which might be due to the fact that having two zinc finger domains
which might be due to the fact that the aggregation propensity of two zinc finger domains
could not be sufficiently compensated by the addition of one solubility
could not be sufficiently compensated by the addition of a single solubility
domain.</span><!-- TU SE KONEA GLAVNO BESEDILO NA STRANI -->
domain.</span><!-- TU SE KONEA GLAVNO BESEDILO NA STRANI -->
</div>
</div>
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     <tr>
     <tr>
       <td style="text-align: right;"><a
       <td style="text-align: right;"><a
  href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/desifriranjebrezfooter"><img
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  src="http://openwetware.org/images/d/d3/Previousblue.JPG"></a></td>
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  href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/desifriranjebrezfooter"><img
  href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultstightbindingzfp"><img
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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 ZFPs</span></big></big></big></big><br>

<br><br> <table 

style="margin: 3px 0px 20px 20px; width: 444px; height: 50px; float: right;"
border="0" cellpadding="0" cellspacing="0">
 <tbody>
   <tr>
     <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 7:

Three dimensional 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 (orange and blue).</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 <a href="http://2010.igem.org/Team:Slovenia">iGEM2010 competition</a> used zinc fingers for positioning proteins on the linear DNA within bacterial cells and did not face the problem of solubility and propensity of ZFPs to aggregate <em>in vitro</em>. 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 a tendency to aggregate with highly soluble protein often results in rescuing the "misbehaved 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 however not straightforward and without problems) is described in more detail in <a href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/methplasmidconst">Plasmid construction</a> 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 added solubility domains or 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 <a href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultsfunctionalizedzfp">Functionalized ZFPs</a>). </span><br 

style="font-family: Arial;">

<br style="font-family: Arial;"> <span style="font-family: Arial;"> We produced six zinc finger protein chimeras tagged with GST. Three of them, GST-Zif268, GST-sZif268 and GST-PBSII, contained three-finger domains recognizing 9 base pairs of their DNA targets. On <strong>Figure 8</strong> we present the analysis of soluble and insoluble fractions of bacterial lysates containing chimeric ZFPs (detailed methods are described in <a href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/methprotprodandisol">Protein expression</a> of the Methods section). <br> <br> </span><span style="font-family: Arial;">Two of the proteins, GST-Zif268 and GST-sZif268, were also successfully purified by chelating chromatography as shown in <strong>Figure 9</strong> of SDS-PAGE and Western blot analysis.</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 8: 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 (Mr of 38 kDa), GST-sZif (Mr of 38 kDa), GST-PBSII (Mr of 38 kDa). Top: Coomassie stained SDS-PAGE gels. Bottom: Western blot analysis using anti-His<sub>4</sub> antibodies. 

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

Figure 9: Purification of GST-fused three-finger domain containing ZFPs.</span> Each image shows a sample of isolated proteins GST-Zif268 (Mr of 38 kDa) and GST-sZif (Mr of 38 kDa). Top: Coomassie stained SDS-PAGE gels. Bottom: western blot analysis using anti-His<sub>4</sub> antibodies. 

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

</table> <br> <span style="font-family: Arial;"> Additionally, we isolated three chimeric proteins 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 10. All of them were successfully purified as shown in Figure 11. </span><br style="font-family: Arial;"> <table style="text-align: left; width: 100%;" border="0" 

cellpadding="8" cellspacing="0">
 <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 10: Bacterial 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 (Mr of approximately 47 kDa), GST-2C7 (Mr of approximately 49 kDa), GST-6F6 (Mr of approximately 48 kDa). Top: Coomassie stained SDS-PAGE gels. Bottom: Western blot analysis using anti-His<sub>4</sub> antibodies. </span></td> 

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

Figure 11: Purification of GST-fused six-finger domains containing ZFPs. </span> Each image shows a sample of isolated proteins GST-AZPA4 (Mr of approximately 47 kDa), GST-2C7 (Mr of approximately 49 kDa) and GST-6F6 (Mr of approximately 48 kDa). Top: Coomassie stained SDS-PAGE gels. Bottom: Western blot analysis using anti-His<sub>4</sub> 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, which can be seen in Figure <strong>12</strong>. </span><br style="font-family: Arial;"> <table style="text-align: left; width: 100%;" border="0" 

cellpadding="8" cellspacing="2">
 <tbody>
   <tr align="center">
     <td><img
style="margin-top: 10px; width: 609px; height: 265px;" alt=""
src="http://openwetware.org/images/e/ec/MBPsolublezadnji.png"></td>
   </tr>
   <tr align="justify">
     <td valign="undefined"><span
style="font-family: Arial;"><span
style="font-weight: bold;">

Figure 12: Production and isolation of MBP-fused ZFPs containing six-finger domains.</span> Left to right: Coomassie stained SDS-PAGE gels of soluble and insoluble fractions of lysates of bacteria producing MBP-6F6 (Mr of approximately 64 kDa) and MBP-2C7 (Mr of approximately 65 kDa); Western blot analysis of MBP-6F6 and MBP-2C7 soluble and insoluble fractions using anti-His<sub>4</sub> antibodies; Coomassie stained SDS-PAGE of isolated MBP-6F6 (Mr of approximately 64 kDa); Coomassie stained SDS-PAGE of isolated MBP-6F6 (Mr of approximately 65 kDa). 

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

</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. </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 chimeric proteins which were designed for the vertical DNA origami stacking using protein tethers. In this case, solubility was also increased by the addition of MBP domain, nevertheless it was somewhat lower than that for other proteins described in this section, which might be due to the fact that the aggregation propensity of two zinc finger domains could not be sufficiently compensated by the addition of a single 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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