Difference between revisions of "Team:Freiburg/Project/Protein Purification"

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<h1 class="sectionedit1">The biochemistry behind protein purification</h1>
 
<h1 class="sectionedit1">The biochemistry behind protein purification</h1>
 
<p>
 
<p>
Cell-free protein expression is a versatile tool for producing proteins at the location where they are needed, in a purity that is not reached with conventional methods. But as this technique is susceptible to changes in the experimental conditions they have to be optimized thorougly. So we decided to use conventional expression in <i>Escherichia coli</i> to reliably get bigger amounts of protein for testing and optimizing additional to cell-free protein expression.
+
Cell-free protein expression is a versatile tool for producing proteins at the location where they are needed, in a purity that is not reached with conventional methods. But as this technique is susceptible to changes in the experimental conditions they have to be optimized thoroughly. So we decided to use conventional expression in <i>Escherichia coli</i> to reliably get higher yields of protein for testing and optimizing additional to cell-free protein expression.
 
</p>
 
</p>
  
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<p>
 
<p>
The genetic information for our antigens was transformed into the bacteria we used (see: <a class="urlextern" href="https://2015.igem.org/Team:Freiburg/Project/Coli_Strains"target="_blank">strains we used</a>). As production of alien proteins (heterologous expression) causes severe stress in the cells, it is reduced by use of the lac-repression system. Only after addition of the non-degradable allolactose analogon IPTG (Isopropylthiogalactopyranosid) the lac-repressor dissociates from its binding site and gives way for transcription. To achieve fast expression the viral T7 RNA polymerase is used instead of endogenous polymerases.
+
The genetic information for our antigens was transformed into the bacteria we used (see: <a class="urlextern" href="https://2015.igem.org/Team:Freiburg/Project/Coli_Strains"target="_blank">strains we used</a>). As production of alien proteins (heterologous expression) can cause severe stress in the cells, the expression level is controlled by using the lac-repression system. Only after addition of the non-degradable allolactose analogon IPTG (Isopropylthiogalactopyranosid) the lac-repressor dissociates from its binding site and gives way for transcription. To achieve fast expression the viral T7 RNA polymerase is used instead of endogenous polymerases.
  
To yield optimal protein concentrations each expression system has first to be optimized in terms of temperature and IPTG-concentration.  
+
To yield optimal protein concentrations each expression system has first to be optimized in terms of temperature and IPTG-concentration as both factors strongly influence yield and solubility of proteins produced.
  
 
This is done by starting with small scales and testing a series of conditions while the yield is quantified by SDS-PAGE. Once the optimal condition is found, the scale is increased and the proteins are purified to a purity that is suitable for our experiments.
 
This is done by starting with small scales and testing a series of conditions while the yield is quantified by SDS-PAGE. Once the optimal condition is found, the scale is increased and the proteins are purified to a purity that is suitable for our experiments.
  
In our basic workflow cells are first grown to an optical density of 0.5 before they are induced by IPTG (figure 1). This is done to ascertain that there are enough cells to overcome the stress caused by induction. After a certain expression time (2, 4 or 8 hours) cells are harvested by centrifugation and can be stored by freezing <sup><a class="fn_top" href="#fn__1" id="fnt__1" name="fnt__1">1)</a></sup><sup><a class="fn_top" href="#fn__2" id="fnt__2" name="fnt__2">2)</a></sup>.
+
In our basic work-flow cells are first grown to an optical density of 0.5 before they are induced by IPTG (figure 1). This is done to ascertain that there are enough cells to overcome the stress caused by induction. After a certain expression time (2, 4 or 8 hours) cells are harvested by centrifugation and can be stored by freezing <sup><a class="fn_top" href="#fn__1" id="fnt__1" name="fnt__1">1)</a></sup><sup><a class="fn_top" href="#fn__2" id="fnt__2" name="fnt__2">2)</a></sup>.
 
</p>
 
</p>
  
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<h2 class="sectionedit3">Getting protein out of cells</h2>
 
<h2 class="sectionedit3">Getting protein out of cells</h2>
 
<p>
 
<p>
When cells are destroyed and proteins are released into solution they are instantly degraded by proteases. These proteins do not get in contact with the target protein in the intact cell. But in the disrupted cell the degradation has to be prevented by addition of inhibitors. We used <a class="urlextern" href="https://2015.igem.org/Team:Freiburg/Glossary"target="_blank">PMSF</a>, a small organic molecule that specifically inhibits serine proteases, by adding it to the solution just before cell lysis <sup><a class="fn_top" href="#fn__3" id="fnt__3" name="fnt__3">3)</a></sup>. Cell lysis was done by sonification, the use of ultrasonic pressure to mechanically destroy the bacterial cell wall. To remove most of the cell debris, the crude lysate was first centrifuged, the supernatant was transferred into a new tube and a second centrifugation was carried out. For this second step higher accelerations were needed to pellet cell organelles and membranes. Most of the target proteins can be found in the pellet (inclusion bodies) as well as in the supernatant (soluble fraction) but only the soluble fraction was used for further purification.
+
When cells are destroyed and proteins are released into solution they are instantly degraded by proteases. These proteins do not get in contact with the target protein in the intact cell. But in the disrupted cell the degradation has to be prevented by addition of inhibitors. We used <a class="urlextern" href="https://2015.igem.org/Team:Freiburg/Glossary"target="_blank">PMSF</a>, a small organic molecule that specifically inhibits serine proteases, by adding it to the solution just prior to cell lysis <sup><a class="fn_top" href="#fn__3" id="fnt__3" name="fnt__3">3)</a></sup>. Cell lysis was performed by sonification, the use of ultrasonic pressure to mechanically destroy the bacterial cell wall. To remove most of the cell debris, the crude lysate was first centrifuged, the supernatant was transferred into a new tube and a second centrifugation was applied. To pellet organelles and membranes, higher centrifugal forces had to be applied. The target proteins can be found divided between the pellet (inclusion bodies) and the supernatant (soluble fraction). The soluble fraction contains natively folded proteins and was used for further purification.  
 
</p>
 
</p>
  
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<h2 class="sectionedit4">Create a useable protein solution</h2>
 
<h2 class="sectionedit4">Create a useable protein solution</h2>
  
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<p>
 
<p>
Even after centrifugation the protein remains far from pure. To specifically address the protein of interest an affinity purification method is best suited. Therefore, a decahistidine tag is fused to the protein whose interaction with divalent cations as nickel allows for selective binding. The protein solution then is pipetted onto an agarose matrix with nickel ions coordinated to NTA-groups. Several washing steps remove unspecific binding of other proteins and the antigen is finally eluted with imidazole that competes for the binding to the cation (figure 2).  
+
Even after centrifugation the protein remains far from pure. To specifically enrich the protein of interest, many different affinity-based methods have been developed. Therefore, a decahistidine tag is fused to the protein. The interaction of these amino-residues with divalent cations as nickel allows for selective binding to a stationary phase. The protein solution then is pipetted onto an agarose matrix with nickel ions coordinated to NTA-groups. Several washing steps remove unspecific binding of other proteins and the antigen is finally eluted with imidazole that competes for the binding to the cation (figure 2).  
Despite having a much purer antigen solution, the eluate contains a high concentration of imidazole (figure 3).  
+
After elution the protein is much purer, but also the concentration of imidazole, used for elution, is strongly increased (figure 3).  
 
</p>
 
</p>
 
<p>
 
<p>
For our project, the expressed proteins are supposed to be immobilized on a glass slide. Since the expressed proteins are fused to a His-tag, whilst the glass surface bears the respective catchers for the tags like a Ni-NTA-modified surface, we are able to specifically bind the antigen onto the slide. To restore its binding capacities, the imidazole has to be removed first by using desalting columns. We used molecular weight cutoff spin columns, that retract all molecules above a specific molecular weight and thus let pass the small salt ions. With this system the elution buffer can gradually be exchanged against an imidazole free spotting buffer <sup><a class="fn_top" href="#fn__1" id="fnt__1" name="fnt__1">1)</a></sup><sup><a class="fn_top" href="#fn__4" id="fnt__4" name="fnt__4">4)</a></sup>.
+
For our project, the expressed proteins are supposed to be immobilized on a glass slide. Since the expressed proteins are fused to a His-tag, whilst the glass surface bears the respective catchers for the tags like a Ni-NTA-modified surface, we are able to specifically bind the antigen onto the slide. To restore its binding capacities, the imidazole has to be removed first by using desalting columns. We used molecular weight cut-off spin columns, that retract all molecules above a specific molecular weight and thus let pass smaller molecules including the small salt ions. With this system the elution buffer can gradually be exchanged against an imidazole free spotting buffer <sup><a class="fn_top" href="#fn__1" id="fnt__1" name="fnt__1">1)</a></sup><sup><a class="fn_top" href="#fn__4" id="fnt__4" name="fnt__4">4)</a></sup>.
 
</p>
 
</p>
 
<div style="margin: 0 10%;">
 
<div style="margin: 0 10%;">
<img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/d/df/Freiburg_versuch2-04.jpg" width=100%/><p><strong> Figure 3: Schematical overview of protein purification using gravity flow columns.</strong> </p>
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<img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/d/df/Freiburg_versuch2-04.jpg" width=100%/><p><strong> Figure 3: Schematic overview of protein purification using gravity flow columns.</strong> </p>
 
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</div>
 
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<!-- EDIT4 SECTION "Create a useable protein solution" [3403-] -->

Revision as of 14:57, 17 September 2015

""

The biochemistry behind protein purification

Cell-free protein expression is a versatile tool for producing proteins at the location where they are needed, in a purity that is not reached with conventional methods. But as this technique is susceptible to changes in the experimental conditions they have to be optimized thoroughly. So we decided to use conventional expression in Escherichia coli to reliably get higher yields of protein for testing and optimizing additional to cell-free protein expression.

Using the natural expression machinery: Growing cells

Figure 1: Schematic overview of protein overexpression.Transformed E. coli are grown to an OD of 0.5 and then induced with IPTG. After a certain time the cells are harvested and can be stored frozen.

The genetic information for our antigens was transformed into the bacteria we used (see: strains we used). As production of alien proteins (heterologous expression) can cause severe stress in the cells, the expression level is controlled by using the lac-repression system. Only after addition of the non-degradable allolactose analogon IPTG (Isopropylthiogalactopyranosid) the lac-repressor dissociates from its binding site and gives way for transcription. To achieve fast expression the viral T7 RNA polymerase is used instead of endogenous polymerases. To yield optimal protein concentrations each expression system has first to be optimized in terms of temperature and IPTG-concentration as both factors strongly influence yield and solubility of proteins produced. This is done by starting with small scales and testing a series of conditions while the yield is quantified by SDS-PAGE. Once the optimal condition is found, the scale is increased and the proteins are purified to a purity that is suitable for our experiments. In our basic work-flow cells are first grown to an optical density of 0.5 before they are induced by IPTG (figure 1). This is done to ascertain that there are enough cells to overcome the stress caused by induction. After a certain expression time (2, 4 or 8 hours) cells are harvested by centrifugation and can be stored by freezing 1)2).

Getting protein out of cells

When cells are destroyed and proteins are released into solution they are instantly degraded by proteases. These proteins do not get in contact with the target protein in the intact cell. But in the disrupted cell the degradation has to be prevented by addition of inhibitors. We used PMSF, a small organic molecule that specifically inhibits serine proteases, by adding it to the solution just prior to cell lysis 3). Cell lysis was performed by sonification, the use of ultrasonic pressure to mechanically destroy the bacterial cell wall. To remove most of the cell debris, the crude lysate was first centrifuged, the supernatant was transferred into a new tube and a second centrifugation was applied. To pellet organelles and membranes, higher centrifugal forces had to be applied. The target proteins can be found divided between the pellet (inclusion bodies) and the supernatant (soluble fraction). The soluble fraction contains natively folded proteins and was used for further purification.

Create a useable protein solution

Figure 2: Comparison of the structure of Imidazole and Histidine.

Even after centrifugation the protein remains far from pure. To specifically enrich the protein of interest, many different affinity-based methods have been developed. Therefore, a decahistidine tag is fused to the protein. The interaction of these amino-residues with divalent cations as nickel allows for selective binding to a stationary phase. The protein solution then is pipetted onto an agarose matrix with nickel ions coordinated to NTA-groups. Several washing steps remove unspecific binding of other proteins and the antigen is finally eluted with imidazole that competes for the binding to the cation (figure 2). After elution the protein is much purer, but also the concentration of imidazole, used for elution, is strongly increased (figure 3).

For our project, the expressed proteins are supposed to be immobilized on a glass slide. Since the expressed proteins are fused to a His-tag, whilst the glass surface bears the respective catchers for the tags like a Ni-NTA-modified surface, we are able to specifically bind the antigen onto the slide. To restore its binding capacities, the imidazole has to be removed first by using desalting columns. We used molecular weight cut-off spin columns, that retract all molecules above a specific molecular weight and thus let pass smaller molecules including the small salt ions. With this system the elution buffer can gradually be exchanged against an imidazole free spotting buffer 1)4).

Figure 3: Schematic overview of protein purification using gravity flow columns.