Team:Tuebingen/Project/SyntheticPeptides

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<h1>Synthetic Peptides</h1>
<h1>Synthetic Peptides</h1>
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<p>
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Two of the previously described protein tags (SpyTag and Intein) can be used with synthetic peptides for covalent immobilization. First the peptides need to be bound to a matrix. In a second step they interact specifically with their corresponding protein-tag, thereby forming a covalent bond. Afterwards, the matrix can be loaded onto columns in order to modify solutions passing it. The covalent binding of tag and peptide ensures that the protein can not be washed from the column and is essential for systems, where the immobilized protein should not be detectable in the product solution.
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</p>
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<p>
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The peptides consist of a tag-specific and an immobilization region. The tag-specific region is specific for the reactivity of the peptide with its corresponding protein tag.
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</p>
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<p>
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The synthetic intein consists of 6 amino acids (GVFVHN) and was for purposes of better steric accessibility extended at the C-terminus by the two amino acids G and A generating the desired active GVFVHNGA peptide of the intein. This synthetic peptide interacts with the intein part of our fusion proteins which leads to an event similar to splicing. For a detailed mechanism visit the wiki-page concerning <a href="https://2014.igem.org/Team:Tuebingen/Project/AffinityTags">Protein-Tags</a>.
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</p>
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<p>
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The synthetic SpyTag consists of 13 amino acids. The sequence did not require additional modification since it was proven by <a href="http://www.pnas.org/content/109/12/E690.abstract">Zakeri et al. in 2011</a> to be suitable for our needs. The resulting functional sequence of the SpyTag is therefore AHIVMVDAYKPTK. The reactivity of this part is mediated by the aspartic acid at position 7, which forms an <i>iso</i>-peptide-bond with a corresponding lysine residue in the SpyCatcher.
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  </p>
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<p>
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The immobilization-region is designed for the binding to the solid support and detection of the peptides. It is located at the C-terminus of the tag-specific region and consists of aminocaproic acid (&epsilon;-AHX or Ahx), lysine and cysteine. The &epsilon;-amino group of the lysine is coupled to a carboxyfluorescein to enable easy detection the peptide. In this way also the detection of our fusion proteins by fluorescence labeling is achievable with low effort. The &epsilon;-AHX functions as spacer between the variable region and the fluorescein to ensure that the reactivity is not decreased due to steric hinderance by the carboxyfluorescein. The cysteine at the C-terminus is used for immobilization on commercial available SulfoLink-Resins or other thio-reactive matrices. The sequence of the immobilization-region of our peptides is therefore AhxK*C, for K* as the labelled lysine.
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</p>
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<p>Two of the previously described protein tags (SpyTag and Intein) can be used with synthetic peptides for covalent immobilization. The peptides can be bound to a matrix and interact with their specific tag, thereby forming a covalent bond. Afterwards, the matrix can be loaded onto columns in order to modify solutions passing it. The strong covalent interaction between tag and peptide is essential for systems, where the immobilized protein should not be detectable in the product solution.</p>
 
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<p>The peptides consist of a variable and an invariable region. The variable region is specific for the reactivity of the peptide with its corresponding protein tag.</p>
 
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<p>The synthetic intein consists of 6 amino acids (GVFVHN) and was for purposes of better steric accessibility extended at the C-terminus by the two amino acids G and A in that way generating the desired active GVFVHNGA peptide of the intein. This synthetic peptide interacts with the intein part fused to the protein residue and leads to an event similar to splicing. For a detailed mechanism visit the wiki-page concerning <a href="https://2014.igem.org/Team:Tuebingen/Project/ProteinTags">Protein-Tags</a>.</p>
 
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<p>The synthetic SpyTag consists of 13 amino acids. The sequence did not require additional modification since it was proven by <a href="http://www.pnas.org/content/109/12/E690.abstract">Zakeri et al. in 2011</a> to be the best suitable one for our needs. The resulting functional sequence of the SpyTag is therefore AHIVMVDAYKPTK. The reactivity of this part is mediated by the aspartic acid at position 7, which forms an <i>iso</i>-peptide-bond with a corresponding lysine residue in the SpyCatcher.</p>
 
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<p>The invariable region is designed for immobilization and detection of the peptides. It is located at the C-terminus of the variable region and consists of aminocaproic acid (&epsilon;-AHX or Ahx), lysine and cysteine. The &epsilion;-amino group of the lysine is coupled to a carboxyfluorescein to enable an easy way to detect the peptide. Detection of desired proteins by fluorescence labeling is in this way achievable with low effort. The &epsilon;-AHX functions as an place holder between the variable region and the fluorescein to ensure that the reactivity is not influenced due to steric hinderance. The cysteine at the c-terminus is usable for immobilization with commercial available SulfoLink-Resins or other thio-reactive matrices. The sequence of the invariable part is therefore AhxK*C, for K* as the labelled lysine.</p>
 
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<p>In essence, the following sequences result from these considerations:</p>
<p>In essence, the following sequences result from these considerations:</p>
<table>
<table>
   <tr>
   <tr>
     <td>Synthetic Intein:</td>
     <td>Synthetic Intein:</td>
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     <td>GVFVHNGA- AhxK*C</td>
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     <td>&nbsp;GVFVHNGA- AhxK*C</td>
   </tr>
   </tr>
   <tr>
   <tr>
     <td>Synthetic SpyTag:</td>
     <td>Synthetic SpyTag:</td>
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     <td>AHIVMVDAYKPTK- AhxK*C</td>
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     <td>&nbsp;AHIVMVDAYKPTK- AhxK*C</td>
   </tr>
   </tr>
</table>
</table>
<img src="https://static.igem.org/mediawiki/2014/6/6d/Tue2014_Structure_Synth-Intein.jpg">
<img src="https://static.igem.org/mediawiki/2014/6/6d/Tue2014_Structure_Synth-Intein.jpg">
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<p id="picText">Figure 1: Structure of our synthetic intein.</p>
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<p id="picTextCenter">Figure 1: Structure of our synthetic intein.</p>
<p>&nbsp;</p>
<p>&nbsp;</p>
<img src="https://static.igem.org/mediawiki/2014/8/8c/Tue2014_Structure_Synth-Spy.jpg">
<img src="https://static.igem.org/mediawiki/2014/8/8c/Tue2014_Structure_Synth-Spy.jpg">
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<p id="picText">Figure 2: Structure of our synthetic SpyTag.</p>
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<p id="picTextCenter">Figure 2: Structure of our synthetic SpyTag.</p>
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<p>Peptides were obtained by solid-phase synthesis using the Fmoc strategy.<br>
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<p><a href="https://2014.igem.org/Team:Tuebingen/Notebook/Protocols/SyntheticPeptides">Click here in order to have a look at our protocol for the creation of synthetic peptides.</a></p>
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Cleavable TentaGel&reg; R RAM beads from <i>Rapp Polymers</i> was used for synthesis. Deprotection of the Fmoc-protected amino group on the surface of the TentaGel&reg;-Resin occurred twice with 1 % piperidine in dimethylformamide (DMF) for 30 min. Cysteine was activated with 1.2 equivalents of HBTU and added twice in 5-fold excess. The product was washed 5 times in DMF and 2 times in dichloromethane (DCM). Deprotection of the Fmoc-protected amino group of the coupled cysteine was performed like the deprotection of the TentaGel&reg;-Resin. HBTU (1.2 eq.) activated lysine (Fmoc-Lys(Mtt)-OH) was coupled twice in 3-fold excess, with the second coupling taking place overnight. The 4-methyltrityl (Mtt) protection group of the washed product was removed by usage of 1 % trifluoroacetic acid (TFA) in DCM and flow-through method. The now unprotected &epsilon;-amino group of the lysine was coupled twice with 5-fold excess to HBTU activated carboxyfluorescein, whereas the second coupling occurred overnight. After extensive washing and deprotection of the &alpha;-amino group, the resin was transferred into a peptide synthesis automat and processed over two days. Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Ahx-OH were used for this purpose. Additionally added <i>N</i>-methyl-2-pyrrolidone (NMP) to the solvents increased the solubility of the amino acids and prevented precipitation in the machine.</p>
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<p>The processed product was cleaved from the resin and unprotected from their side chain protection groups using a cleavage solution consisting of 670 mg of phenol, 450 &mu;l of water, 450 &mu;l of triisopropylsilyl (TIPS) and 9 ml of TFA. The resulting peptides were precipitated in cold diethyl ether and lyophilized overnight. Purification was executed by high-performance liquid chromatography (HPLC) and the resulting product was checked by mass spectroscopy using electron electrospray ionization (ESI).</p>
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</div>
</div>

Latest revision as of 03:36, 18 October 2014


Synthetic Peptides

Two of the previously described protein tags (SpyTag and Intein) can be used with synthetic peptides for covalent immobilization. First the peptides need to be bound to a matrix. In a second step they interact specifically with their corresponding protein-tag, thereby forming a covalent bond. Afterwards, the matrix can be loaded onto columns in order to modify solutions passing it. The covalent binding of tag and peptide ensures that the protein can not be washed from the column and is essential for systems, where the immobilized protein should not be detectable in the product solution.

The peptides consist of a tag-specific and an immobilization region. The tag-specific region is specific for the reactivity of the peptide with its corresponding protein tag.

The synthetic intein consists of 6 amino acids (GVFVHN) and was for purposes of better steric accessibility extended at the C-terminus by the two amino acids G and A generating the desired active GVFVHNGA peptide of the intein. This synthetic peptide interacts with the intein part of our fusion proteins which leads to an event similar to splicing. For a detailed mechanism visit the wiki-page concerning Protein-Tags.

The synthetic SpyTag consists of 13 amino acids. The sequence did not require additional modification since it was proven by Zakeri et al. in 2011 to be suitable for our needs. The resulting functional sequence of the SpyTag is therefore AHIVMVDAYKPTK. The reactivity of this part is mediated by the aspartic acid at position 7, which forms an iso-peptide-bond with a corresponding lysine residue in the SpyCatcher.

The immobilization-region is designed for the binding to the solid support and detection of the peptides. It is located at the C-terminus of the tag-specific region and consists of aminocaproic acid (ε-AHX or Ahx), lysine and cysteine. The ε-amino group of the lysine is coupled to a carboxyfluorescein to enable easy detection the peptide. In this way also the detection of our fusion proteins by fluorescence labeling is achievable with low effort. The ε-AHX functions as spacer between the variable region and the fluorescein to ensure that the reactivity is not decreased due to steric hinderance by the carboxyfluorescein. The cysteine at the C-terminus is used for immobilization on commercial available SulfoLink-Resins or other thio-reactive matrices. The sequence of the immobilization-region of our peptides is therefore AhxK*C, for K* as the labelled lysine.

In essence, the following sequences result from these considerations:

Synthetic Intein:  GVFVHNGA- AhxK*C
Synthetic SpyTag:  AHIVMVDAYKPTK- AhxK*C

Figure 1: Structure of our synthetic intein.

 

Figure 2: Structure of our synthetic SpyTag.

Click here in order to have a look at our protocol for the creation of synthetic peptides.

Retrieved from "http://2014.igem.org/Team:Tuebingen/Project/SyntheticPeptides"