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<div class="content"><h2> Short summary </h2> | <div class="content"><h2> Short summary </h2> | ||
<article> | <article> | ||
− | The | + | The non-canonical amino acid (ncAA) 2-nitrophenylalanine (2-NPA) has the special property to induce a cleavage of the peptide backbone when irradiated with light of a wavelength of > 300 nm. To demonstrate the usage of 2-NPA, we designed a fusion protein of the green fluorescent protein (GFP) and streptavidin connected by a glycine-glycine-serine-linker. The streptavidin compound will form a stable and highly specific non-covalent bond to biotin, so that the fusion protein can easily be immobilized on any biotinylated surface. The GFP is used as a fluorescence tag so that the fusion protein can easily be identified through its fluorescent properties. The immobilized fusion protein can then be irradiated with light to induce the cleavage of the peptide backbone and elute the target protein |
</article> | </article> | ||
</div> | </div> | ||
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<div class="figure medium"> | <div class="figure medium"> | ||
<img class="figure image" src="https://static.igem.org/mediawiki/2017/f/f1/T--Bielefeld-CeBiTec--photo_cleavage_mechanism.jpg"> | <img class="figure image" src="https://static.igem.org/mediawiki/2017/f/f1/T--Bielefeld-CeBiTec--photo_cleavage_mechanism.jpg"> | ||
− | <p class="figure subtitle"><b>Figure 1a: Reaction scheme of the proposed mechanism of photocleavage reaction by Peters et al. | + | <p class="figure subtitle"><b>Figure 1a:</b> Reaction scheme of the proposed mechanism of photocleavage reaction by Peters et al. |
</div> | </div> | ||
<div class="figure medium"> | <div class="figure medium"> | ||
<img class="figure image" src="https://static.igem.org/mediawiki/2017/8/83/T--Bielefeld-CeBiTec--YKE_Photolysis_one.gif"> | <img class="figure image" src="https://static.igem.org/mediawiki/2017/8/83/T--Bielefeld-CeBiTec--YKE_Photolysis_one.gif"> | ||
− | <p class="figure subtitle"><b>Figure 1b: Animation of the proposed mechanism of photocleavage reaction by Peters et al. | + | <p class="figure subtitle"><b>Figure 1b:</b> Animation of the proposed mechanism of photocleavage reaction by Peters et al. |
</div> | </div> | ||
<h4> Characteristics of the ncAA </h4> | <h4> Characteristics of the ncAA </h4> | ||
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<div class="figure medium"> | <div class="figure medium"> | ||
<img class="figure image" src="https://static.igem.org/mediawiki/2017/c/ca/T--Bielefeld-CeBiTec--YKE_2NPA-sec.svg"> | <img class="figure image" src="https://static.igem.org/mediawiki/2017/c/ca/T--Bielefeld-CeBiTec--YKE_2NPA-sec.svg"> | ||
− | <p class="figure subtitle"><b>Figure 2: Structure of 2-Nitrophenylalanine | + | <p class="figure subtitle"><b>Figure 2:</b> Structure of 2-Nitrophenylalanine. |
</div> | </div> | ||
</div> | </div> | ||
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<div class="figure medium"> | <div class="figure medium"> | ||
<img class="figure image" src="https://static.igem.org/mediawiki/2017/d/df/T--Bielefeld-CeBiTec--YKE_light_induced_elution_concept.png"> | <img class="figure image" src="https://static.igem.org/mediawiki/2017/d/df/T--Bielefeld-CeBiTec--YKE_light_induced_elution_concept.png"> | ||
− | <p class="figure subtitle"><b>Figure | + | <p class="figure subtitle"><b>Figure 3:</b> Overview of the light induced elution process with our fusion protein containing 2-NPA in the protein purification column. |
</div> | </div> | ||
<article> | <article> | ||
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<div class="figure medium"> | <div class="figure medium"> | ||
<img class="figure image" src="https://static.igem.org/mediawiki/2017/e/ee/T--Bielefeld-CeBiTec--YKE_GFP_structure_one.png"> | <img class="figure image" src="https://static.igem.org/mediawiki/2017/e/ee/T--Bielefeld-CeBiTec--YKE_GFP_structure_one.png"> | ||
− | <p class="figure subtitle"><b>Figure | + | <p class="figure subtitle"><b>Figure 4:</b> Three perspectives of the photoproduct of the tetrameric wild type Aequorea victoria green fluorescent protein from rcsb.org. |
</div> | </div> | ||
<article> | <article> | ||
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<div class="figure small"> | <div class="figure small"> | ||
<img class="figure image" src="https://static.igem.org/mediawiki/2017/3/3a/T--Bielefeld-CeBiTec--fluorescent_proteins_variants.jpg"> | <img class="figure image" src="https://static.igem.org/mediawiki/2017/3/3a/T--Bielefeld-CeBiTec--fluorescent_proteins_variants.jpg"> | ||
− | <p class="figure subtitle"><b>Figure | + | <p class="figure subtitle"><b>Figure 5:</b> Three perspectives of the photoproduct of the tetrameric wild type Aequorea victoria green fluorescent protein from rcsb.org. |
</div> | </div> | ||
<h4> Streptavidin</h4> | <h4> Streptavidin</h4> | ||
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<div class="figure medium"> | <div class="figure medium"> | ||
<img class="figure image" src="https://static.igem.org/mediawiki/2017/b/b7/T--Bielefeld-CeBiTec--YKE_streptavidin_structure_one.png"> | <img class="figure image" src="https://static.igem.org/mediawiki/2017/b/b7/T--Bielefeld-CeBiTec--YKE_streptavidin_structure_one.png"> | ||
− | <p class="figure subtitle"><b>Figure | + | <p class="figure subtitle"><b>Figure 6:</b> Three perspectives of the structure of a wild type streptavidin tetramer in complex with biotin from rcsb.org. |
</div> | </div> | ||
<article> | <article> | ||
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<div class="figure small"> | <div class="figure small"> | ||
<img class="figure image" src="https://static.igem.org/mediawiki/2017/0/0a/T--Bielefeld-CeBiTec--YKE_fusion_protein_structure_one.png"> | <img class="figure image" src="https://static.igem.org/mediawiki/2017/0/0a/T--Bielefeld-CeBiTec--YKE_fusion_protein_structure_one.png"> | ||
− | <p class="figure subtitle"><b>Figure | + | <p class="figure subtitle"><b>Figure 7:</b> Fusion protein of EGFP and Cytochrome b562 by rcsb.org. |
</div> | </div> | ||
<article> | <article> | ||
− | In the design process of recombinant fusion proteins, linkers are very important, as they can increase the stability, bioactivity, and expression yield of the fusion protein (Chen et al., 2013). Additionally, a direct fusion of functional domains without a linker can lead to misfolding and other undesirable properties. Linker sequences can be received from natural multi-domain proteins or be rationally designed depending on the desired characteristics. They are mainly classified in three different groups: flexible linkers, rigid linkers, and cleavable linkers (see figure | + | In the design process of recombinant fusion proteins, linkers are very important, as they can increase the stability, bioactivity, and expression yield of the fusion protein (Chen et al., 2013). Additionally, a direct fusion of functional domains without a linker can lead to misfolding and other undesirable properties. Linker sequences can be received from natural multi-domain proteins or be rationally designed depending on the desired characteristics. They are mainly classified in three different groups: flexible linkers, rigid linkers, and cleavable linkers (see figure 8). Furthermore they can be classified as small (approximately five amino acids), medium (approximately ten) or large linkers (approximately 20 to 28 amino acids) (Weber et al., 1989). |
</article> | </article> | ||
<div class="figure medium"> | <div class="figure medium"> | ||
<img class="figure image" src="https://static.igem.org/mediawiki/2017/2/2f/T--Bielefeld-CeBiTec--YKE_linkers_new.png"> | <img class="figure image" src="https://static.igem.org/mediawiki/2017/2/2f/T--Bielefeld-CeBiTec--YKE_linkers_new.png"> | ||
− | <p class="figure subtitle"><b>Figure | + | <p class="figure subtitle"><b>Figure 8:</b> Three groups of protein linkers. Left: flexible, middle: rigid, right: cleavable. |
</div> | </div> | ||
<article> | <article> |
Revision as of 15:24, 28 October 2017
![](https://static.igem.org/mediawiki/2017/7/74/T--Bielefeld-CeBiTec--title-img-centrifuge.jpg)
Short summary
Photolysis of peptide chains
Explanation of the ncAA
![](https://static.igem.org/mediawiki/2017/f/f1/T--Bielefeld-CeBiTec--photo_cleavage_mechanism.jpg)
Figure 1a: Reaction scheme of the proposed mechanism of photocleavage reaction by Peters et al.
![](https://static.igem.org/mediawiki/2017/8/83/T--Bielefeld-CeBiTec--YKE_Photolysis_one.gif)
Figure 1b: Animation of the proposed mechanism of photocleavage reaction by Peters et al.
Characteristics of the ncAA
- Name: 2-Nitro-L-phenylalanine
- Short: 2-NPA
- CAS: 19883-75-1
- MW: 210.19
- Storage: 2-8°C
- Source: apolloscientific
- Prize: 5g - £205.00
- Function: induces a cleavage of the peptide backbone when radiated with ʎ=365nm
Figure 2: Structure of 2-Nitrophenylalanine.
Theoretical basis
Light-induced elution
![](https://static.igem.org/mediawiki/2017/d/df/T--Bielefeld-CeBiTec--YKE_light_induced_elution_concept.png)
Figure 3: Overview of the light induced elution process with our fusion protein containing 2-NPA in the protein purification column.
We hope that the fusion protein in unfiltered cell lysate will bind strong and specifically to the purification column with biotinylated glass slides, so that the other proteins and cell fragments can be easily washed away. We then want to irradiate the slides with light of 395 nm wave length to detect the GFP and prove the binding efficiency of the streptavidin and the functionality of the selected linker. Afterwards, we want to irradiate the column with UV-light of 365 nm wave length to induce the photocleavage of the 2-NPA. In the following elution step the GFP will be eluted while other proteins that were bound unspecific to the biotinylated surface should not be effected by the irradiation and retain on the column. The elution of the GFP can then also be detected as well as the fluorescence of the eluate.
After using the purification column it should be easily regenerated by simply washing it with SDS-solution. The SDS will denaturate the streptavidin with the linker and the other proteins bound to the column so that they will lose their binding affinity to the biotin and be washed off the glass slides. The biotin itself should not be influenced by the SDS-solution so that the glass slides will still be usable for many purification steps.
To implement all this, we started the development of a purification column, containing the needed biotinylated surfaces and an LED-panel that is able to radiate the needed UV-light with a wave length of 365 nm.
Green fluorescent protein: GFP
![](https://static.igem.org/mediawiki/2017/e/ee/T--Bielefeld-CeBiTec--YKE_GFP_structure_one.png)
Figure 4: Three perspectives of the photoproduct of the tetrameric wild type Aequorea victoria green fluorescent protein from rcsb.org.
![](https://static.igem.org/mediawiki/2017/3/3a/T--Bielefeld-CeBiTec--fluorescent_proteins_variants.jpg)
Figure 5: Three perspectives of the photoproduct of the tetrameric wild type Aequorea victoria green fluorescent protein from rcsb.org.
Streptavidin
![](https://static.igem.org/mediawiki/2017/b/b7/T--Bielefeld-CeBiTec--YKE_streptavidin_structure_one.png)
Figure 6: Three perspectives of the structure of a wild type streptavidin tetramer in complex with biotin from rcsb.org.
Fusion Proteins
![](https://static.igem.org/mediawiki/2017/0/0a/T--Bielefeld-CeBiTec--YKE_fusion_protein_structure_one.png)
Figure 7: Fusion protein of EGFP and Cytochrome b562 by rcsb.org.
![](https://static.igem.org/mediawiki/2017/2/2f/T--Bielefeld-CeBiTec--YKE_linkers_new.png)
Figure 8: Three groups of protein linkers. Left: flexible, middle: rigid, right: cleavable.
References
Peters, F.B., Brock, A., Wang, J., and Schultz, P.G. (2009). Photocleavage of the Polypeptide Backbone by 2-NitrophenylalaninePeters. Chem. Biol. 16: 148–152.Roger Y. Tsien (1998). The Green Fluorescent Protein. Annu. Rev. Biochem. 1998. 67:509–44.
Xiaoying Chen, Jennica Zaro, and Wei-Chiang Shen (2013). Fusion Protein Linkers: Property, Design and Functionality. Adv Drug Deliv Rev. 65(10): 1357–1369.
Patricia C. Weber, D. H. Ohlendorf, J. J. Wendoloski and F. R. Salemme (1989). Structural Origins of High-Affinity Biotin Binding to Streptavidin. Science. 243: 85-88.
Lichty, J.J., Malecki, J.L., Agnew, H.D., Michelson-horowitz, D.J., and Tan, S. (2005). Comparison of affinity tags for protein purification. 41: 98–105.