Difference between revisions of "Team:Bielefeld-CeBiTec/Project/toolbox/fusing"

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<img class="figure image" src="https://static.igem.org/mediawiki/2017/2/22/T--Bielefeld-CeBiTec--27-08-17-CL_structure.png">
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<p class="figure subtitle"><b>Figure 6: Structure of CL.</b><br>Name: N<sup>ε</sup>-L-cysteinyl-L-lysine<br>
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<p class="figure subtitle"><b>Figure 3: Structure of CL.</b><br>Name: N<sup>ε</sup>-L-cysteinyl-L-lysine<br>
 
Molecular Weight: 249.33 g mol<sup>-1</sup><br>
 
Molecular Weight: 249.33 g mol<sup>-1</sup><br>
 
Storage: -20 – 4 °C<br>
 
Storage: -20 – 4 °C<br>
 
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<h3>N<sup>γ</sup> 2 cyanobenzothiazol-6-yl-L-asparagine</h3>
 
<h3>N<sup>γ</sup> 2 cyanobenzothiazol-6-yl-L-asparagine</h3>
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<p class="figure subtitle"><b>Figure 7: Structure of CBT-Asp.</b><br>Name: N<sup>γ</sup>-2-cyanobenzothiazol-6-yl-L-asparagine<br>
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<p class="figure subtitle"><b>Figure 4: Structure of CBT-Asp.</b><br>Name: N<sup>γ</sup>-2-cyanobenzothiazol-6-yl-L-asparagine<br>
 
Molecular Weight: 290.30 g mol<sup>-1</sup><br>
 
Molecular Weight: 290.30 g mol<sup>-1</sup><br>
 
Storage: -20 – 4 °C<br>
 
Storage: -20 – 4 °C<br>
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<h3>Coupling reaction of N-Boc-L-lysine-O-methyl ester and N-Boc-L-cysteine-S-Trt</h3>
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<h3>Silk Elastin like Proteins</h3>
  
 
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Table 1 shows the used quantity of reactants and solvents for both batches.
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This specific binding can improve the stability of SELPs. These are linear polypeptides with repeats of silk and elastin consensus sequences. They show brought application and possibilities in medicine, tissue engineering and industry (Rnjak-Kovacina et al., 2011). The silk consensus sequence is GAGAGS and the elastin consensus sequence is VPAVG. The consensus sequences can interact with each other and are able to form non-covalent hydrogen bonds. This results in a polymer network based on hydrogen bonds with a β-sheet structure. Figure 5 shows the schematic structure of a SELP polymer network.
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<b>Table 1: List of used reactants and solvents for the coupling.</b>
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In both batches, we used the same quantity of reactants and solvents for the coupling reaction.
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/3/36/T--Bielefeld-CeBiTec--27-08-17-Schematic_SELP.png">
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<p class="figure subtitle"><b> </b></p>
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<p class="figure subtitle"><b>Figure 5: Schematic structure of a SELP polymer network.</b><br>Silk consensus sequences are shown in green, elastin consensus sequences are red and the blue lines show the hydrogen bonds of the consensus sequences.</p>
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<p class="figure subtitle"><b>Figure 2: Result of the TLC analysis after the coupling reaction.</b><br>A: N-Boc-L-lysine-O-methyl ester; B: N Boc L cysteine-S-Trt; C: N-Boc-L-lysine-O-methyl ester, N Boc L cysteine-S-Trt and the reaction mixture after the coupling reaction; D: the reaction mixture after the coupling reaction.</p>
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According to the work of Collins et al. (2013), we decided to use a sequence with nine repeats of five repeats of the silk consensus sequence and nine repeats of the elastin consensus sequence (see figure 6).
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The thin layer chromatography (TLC) analysis of the reaction mixture shows that after the coupling reaction no N-Boc-L-lysine-O-methyl ester was left (see figure 2). This indicates that the N-Boc-L-lysine-O-methyl ester completely reacted. The two spots on the top of C and D are the product – the N-Boc-L-lysine[N<sup>ε</sup>-(N-Boc-L-cysteine-S-Trt)]-6-methyl ester (lower spot) – and a byproduct of the reaction (upper spot).
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/0/0e/T--Bielefeld-CeBiTec--27-08-17-SELP_seq_Collins2013.png">
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<p class="figure subtitle"><b>Figure 6: Schematic sequence of the SELP (Collins et al., 2013).</b><br>Silk consensus sequences are green and elastin consensus sequences are red.</p>
 
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By incorporation of CL and CBT-Asp between the silk and the elastin repeats, we receive a strengthened polymer network with covalent bonds (Figure 7).
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/8/8c/T--Bielefeld-CeBiTec--27-08-17-NMR_CL-CBT-Asp1.png">
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<p class="figure subtitle"><b>Figure 3: Nuclear magnetic resonance (NMR) analysis result for the purified reaction mixture after the coupling reaction.</b><br> The signals for the hydrogen bonds of the protecting groups were highlighted because they are characteristic for the estimated product – N-Boc-L-lysine[N<sup>ε</sup>-(N-Boc-L-cysteine-S-Trt)]-6-methyl ester.</p>
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The NMR analysis of the purified reaction mixture of the coupling reaction shows that the hydrogen atoms of all protecting groups are present (see figure 3). The Tritylphenylmethane (Trt) at 7.2 ppm is part of the N-Boc-L-cysteine-S-Trt and the methyl ester at 3.6 ppm is originating from the N-Boc-L-lysine-O-methyl ester. The tert-Butyloxycarbonyl protecting group is part of both educts. In this reaction, should be no protecting groups split off so that you can see here the NMR analysis for N-Boc-L-lysine[N<sup>ε</sup>-(N-Boc-L-cysteine-S-Trt)]-6-methyl ester.
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<h3>Removing the methyl ester of the N-Boc-L-lysine[N<sup>ε</sup>-(N-Boc-L-cysteine-S-Trt)]-6-methyl ester</h3>
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Table 2 shows the used quantity of reactants and solvents for both batches.
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<b>Table 2: List of used reactants and solvents for the reaction to remove methyl ester of the first and the second batch.</b>
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/7/77/T--Bielefeld-CeBiTec--27-08-17-results_Tab1_coupling_reaction.png">
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/9/9e/T--Bielefeld-CeBiTec--27-08-17-SELP_ncAA.png">
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<p class="figure subtitle"><b> </b></p>
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<p class="figure subtitle"><b>Figure 7: Schematic structure of a SELP polymer network.</b></p>
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<p class="figure subtitle"><b>Figure 4: Result of the TLC analysis after removing the methyl ester.</b><br>KC2: the reaction mixture after the coupling reaction; KC3: the reaction mixture after removing the methyl ester.</p>
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<h3>PRe-RDL</h3>
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After removing the methyl ester, the product is more polar than before. The result is that the N-Boc-L-lysine[N<sup>ε</sup>-(N-Boc-L-cysteine-S-Trt)] is not soluble in the EtOAc:PE solution. The dark spot at the TLC plate for the sample KC3 is the N-Boc-L-lysine[N<sup>ε</sup>-(N-Boc-L-cysteine-S-Trt)] and the lighter spot is the removed methyl ester (see figure 4).
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The gene sequence for these SELPs has a high GC content and contains a high number of repeats leading to issues during synthesis. Therefore, Recursive Directional Ligation by Plasmid Reconstruction (PRe RDL) can be applied to address this challenge. Pre-RDL uses three restriction sites of a parent plasmid which contains the gene of interest (goi) and the subsequently ligation of fragments of two different restricted parent plasmids. The first step involves the restriction of a parent plasmid at the 3'-end of the goi and in the backbone. The second step is the restriction of a parent plasmid at the 5'-end of the goi and at the same position of the backbone as in the first step. The final step is the ligation of both generated fragments containing the goi. The result is a plasmid with two copies of the goi (Figure 5). We applied Pre-RDL to build a plasmid with the described SELP gene sequence.
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<h3>Removing tert-Butyloxycarbonyl protecting group (Boc) and Triphenylmethane (Trt) of the N-Boc-L-lysine[N<sup>ε</sup>-(N-Boc-L-cysteine-S-Trt)]</h3>
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Table 3 shows the used quantity of reactants and solvents for both batches.
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<b>Table 3: List of used reactants and solvents for the reaction to remove Boc and Trt of the first and the second batch.</b>
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</article>
 
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/8/8a/T--Bielefeld-CeBiTec--27-08-17-results_Tab3_remove_boc_trt.png">
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/5/59/T--Bielefeld-CeBiTec--27-08-17-PRe-RDL_McDaniel2010.png">
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<p class="figure subtitle"><b> </b></p>
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<p class="figure subtitle"><b>Figure 8: Scheme of the PRe-RDL (McDaniel et al., 2010).</b></p>
 
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/6/6c/T--Bielefeld-CeBiTec--27-08-17-NMR_CL-CBT-Asp2.png">
 
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<p class="figure subtitle"><b>Figure 5: NMR analysis result for the purified N<sup>ε</sup>-L-cysteinyl-L-lysine trifluoroacetatic acid salt.</b><br>All peaks of compounds with hydrogen atoms of the N<sup>ε</sup>-L-cysteinyl-L-lysine were highlighted because they are characteristic for this molecule.</p>
 
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The NMR analysis shows that all estimated hydrogen atoms are present and that the synthesis was successful (see figure 5).
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In the first batch, we got 400 mg of N<sup>ε</sup>-L-cysteinyl-L-lysine trifluoroacetic acid salt and in the second batch 500 mg. This correspond to 0.84 mmol for the first batch and 1.05 mmol for the second batch. This equals to the half of the yield of Nguyen et al. (2011) with 900 mg and 1.89 mmol.
 
 
<br>
 
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Boateng, J., Matthews, K.H., Stevens, H.N.E., Eccleston, G.M., 2008. Wound Healing Dressings and Drug Delivery Systems: A Review. J. Pharm. Sci. 97. doi:10.1002/jps21210
 +
<br>
 +
<br>
 +
Collins, T., Azevedo-silva, J., Costa, A., Branca, F., Machado, R., Casal, M., 2013. Batch production of a silk-elastin-like protein in E . coli BL21 ( DE3 ): key parameters for optimisation. Microb. Cell Fact. 12, 1–16. doi:10.1186/1475-2859-12-21
 +
<br>
 +
<br>
 +
Liang, G., Ren, H., Rao, J., 2010. A biocompatible condensation reaction for controlled assembly of nanostructures in living cells. Nat. Chem. 2, 54–60. doi:10.1038/nchem.480
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<br>
 +
<br>
 +
McDaniel, J.R., Mackay, J.A., Quiroz, F.G., Chilkoti, A., 2010. Recursive Directional Ligation by Plasmid Reconstruction allows Rapid and Seamless Cloning of Oligomeric Genes 11, 944–952. doi:10.1021/bm901387t.Recursive
 
<br>
 
<br>
 
<br>
 
<br>
 
Nguyen, D.P., Elliott, T., Holt, M., Muir, T.W., Chin, J.W., 2011. Genetically Encoded 1,2-Aminothiols Facilitate Rapid and Site-Specific Protein Labeling via a Bio-orthogonal Cyanobenzothiazole Condensation. J. Am. Chem. Soc. 133, 11418–11421. doi:10.1021/ja203111c
 
Nguyen, D.P., Elliott, T., Holt, M., Muir, T.W., Chin, J.W., 2011. Genetically Encoded 1,2-Aminothiols Facilitate Rapid and Site-Specific Protein Labeling via a Bio-orthogonal Cyanobenzothiazole Condensation. J. Am. Chem. Soc. 133, 11418–11421. doi:10.1021/ja203111c
 +
<br>
 +
<br>
 +
Rnjak-Kovacina, J., Daamen, W.F., Pierna, M., Rodríguez-Cabello, J.C., Weiss, A.S., 2011. Elastin Biopolymers. Compr. Biomater. 329–346. doi:http://dx.doi.org/10.1016/B978-0-08-055294-1.00071-4
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</article>
 
</article>
 
 

Revision as of 03:49, 27 August 2017

Fusing

Short summary

Fusing proteins is normally limited to the C- or N-terminus of a protein. The incorporation of non-canonical amino acids that could be fused to each other or to surfaces enables several additional applications. This tool facilitates immobilization of proteins and improved stability of protein polymer networks. Furthermore, they lead to enhanced efficiency of pathways by combining enzymes of one pathway or for any other system where colocalization is beneficial.
As proof of concept, we work on enhanced stability of a protein polymer. This networks can be applied for different applications like modern biomaterials in medicine and industry (Rnjak-Kovacina et al., 2011). The amino acids Nε-L-cysteinyl-L-lysine (CL) and Nγ 2 cyanobenzothiazol 6 yl L asparagine (CBT-Asp) comprise key parts of this tool. Both amino acids can bind specificly to each other resulting in the formation of a covalent bond between their side chains. We plan to use this covalent bond to increase the stability of silk elastin like proteins (SELPs). The strengthened polymer network would be a perfect material to produce biological wound bindings which are very thin and they would be able to interact with the natural tissue matrix (Boateng et al., 2008).

Terminus independent fusion proteins

While terminus dependent binding systems for proteins are already in use, there are only a few systems for terminus independent binding systems. We want to expand the number of those systems. Our aim is to incorporate two non-canonical amino acids, which are able to build a specific bond to each other. According to the synthesis of luciferin for the firefly luciferase of Photinus pyralis, we decided to use the specific binding of 1,2-aminothiols and the cyano group of cyanobenzothiazole (CBT). Figure 1 shows the biosynthesis of luciferin and the mechanism of the binding reaction of CBT and 1,2-aminothiol.

Figure 1: Reaction of the 1,2-aminothiol of cysteine and CBT to luciferin (Liang et al., 2010).

By synthesis of amino acids with side chains containing CBT and a 1,2-aminothiol, polypeptides binding to each other should be produced. These amino acids are CL and CBT-Asp. The binding mechanism of both amino acids are shown in figure 2.

Figure 2: Specific binding reaction of CL and CBT-Asp.

Nε-L-cysteinyl-L-lysine

We synthesized CL in our lab and provide the community with a validated protocol. Currently, we are trying to synthesize CBT-Asp. Details of our synthesis protocol are described in the method section (link to methods).
CL is an amino acid consisting of cysteine and lysine. The cysteine was coupled to the side chain of lysine so that CL contains a free 1,2-aminothiol group (Nguyen et al., 2011). This is an important characteristic for the specific binding between the CL and the CBT-Asp.

Figure 3: Structure of CL.
Name: Nε-L-cysteinyl-L-lysine
Molecular Weight: 249.33 g mol-1
Storage: -20 – 4 °C

Nγ 2 cyanobenzothiazol-6-yl-L-asparagine

CBT-Asp is a completely novel amino acid, which we are synthesizing on our own. to the synthesis is based on coupling the amino group of CBT to the carboxyl group of the side chain of L-asparagine. The cyano group of the cyanobenzothiazol enables the specific binding of the CBT-Asp to 1,2-aminothiols.

Figure 4: Structure of CBT-Asp.
Name: Nγ-2-cyanobenzothiazol-6-yl-L-asparagine
Molecular Weight: 290.30 g mol-1
Storage: -20 – 4 °C

CL is an amino acid consisting of cysteine and lysine. The cysteine was coupled to the side chain of lysine so that CL contains a free 1,2-aminothiol group (Nguyen et al., 2011). This is an important characteristic for the specific binding between the CL and the CBT-Asp.

Silk Elastin like Proteins

This specific binding can improve the stability of SELPs. These are linear polypeptides with repeats of silk and elastin consensus sequences. They show brought application and possibilities in medicine, tissue engineering and industry (Rnjak-Kovacina et al., 2011). The silk consensus sequence is GAGAGS and the elastin consensus sequence is VPAVG. The consensus sequences can interact with each other and are able to form non-covalent hydrogen bonds. This results in a polymer network based on hydrogen bonds with a β-sheet structure. Figure 5 shows the schematic structure of a SELP polymer network.

Figure 5: Schematic structure of a SELP polymer network.
Silk consensus sequences are shown in green, elastin consensus sequences are red and the blue lines show the hydrogen bonds of the consensus sequences.

According to the work of Collins et al. (2013), we decided to use a sequence with nine repeats of five repeats of the silk consensus sequence and nine repeats of the elastin consensus sequence (see figure 6).

Figure 6: Schematic sequence of the SELP (Collins et al., 2013).
Silk consensus sequences are green and elastin consensus sequences are red.

By incorporation of CL and CBT-Asp between the silk and the elastin repeats, we receive a strengthened polymer network with covalent bonds (Figure 7).

Figure 7: Schematic structure of a SELP polymer network.

PRe-RDL

The gene sequence for these SELPs has a high GC content and contains a high number of repeats leading to issues during synthesis. Therefore, Recursive Directional Ligation by Plasmid Reconstruction (PRe RDL) can be applied to address this challenge. Pre-RDL uses three restriction sites of a parent plasmid which contains the gene of interest (goi) and the subsequently ligation of fragments of two different restricted parent plasmids. The first step involves the restriction of a parent plasmid at the 3'-end of the goi and in the backbone. The second step is the restriction of a parent plasmid at the 5'-end of the goi and at the same position of the backbone as in the first step. The final step is the ligation of both generated fragments containing the goi. The result is a plasmid with two copies of the goi (Figure 5). We applied Pre-RDL to build a plasmid with the described SELP gene sequence.

Figure 8: Scheme of the PRe-RDL (McDaniel et al., 2010).



Boateng, J., Matthews, K.H., Stevens, H.N.E., Eccleston, G.M., 2008. Wound Healing Dressings and Drug Delivery Systems: A Review. J. Pharm. Sci. 97. doi:10.1002/jps21210

Collins, T., Azevedo-silva, J., Costa, A., Branca, F., Machado, R., Casal, M., 2013. Batch production of a silk-elastin-like protein in E . coli BL21 ( DE3 ): key parameters for optimisation. Microb. Cell Fact. 12, 1–16. doi:10.1186/1475-2859-12-21

Liang, G., Ren, H., Rao, J., 2010. A biocompatible condensation reaction for controlled assembly of nanostructures in living cells. Nat. Chem. 2, 54–60. doi:10.1038/nchem.480

McDaniel, J.R., Mackay, J.A., Quiroz, F.G., Chilkoti, A., 2010. Recursive Directional Ligation by Plasmid Reconstruction allows Rapid and Seamless Cloning of Oligomeric Genes 11, 944–952. doi:10.1021/bm901387t.Recursive

Nguyen, D.P., Elliott, T., Holt, M., Muir, T.W., Chin, J.W., 2011. Genetically Encoded 1,2-Aminothiols Facilitate Rapid and Site-Specific Protein Labeling via a Bio-orthogonal Cyanobenzothiazole Condensation. J. Am. Chem. Soc. 133, 11418–11421. doi:10.1021/ja203111c

Rnjak-Kovacina, J., Daamen, W.F., Pierna, M., Rodríguez-Cabello, J.C., Weiss, A.S., 2011. Elastin Biopolymers. Compr. Biomater. 329–346. doi:http://dx.doi.org/10.1016/B978-0-08-055294-1.00071-4