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

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                     This system is an alternative procedure of site-specific protein cleavage compared to a cleavage by proteases or chemicals. A big advantage of this light-induced cleavage is, that it can be used almost universally on any purpose. It prevents disadvantages like an undesired cleavage of the target protein by undetected cleavage sides or a denaturation of the protein through the reagents when cleaved chemically. The system has a wide range of possible applications, such as inactivating proteins by cleaving them, activating them by cleaving an inactive pre-protein and releasing an active form, or a combination with other methods as demonstrated in our light-induced elution.

                     The ncAA 2-NPA is able to photochemically cleave the polypeptide backbone by a cinnoline forming reaction based on the photochemistry of (2-nitrophenyl)ethane derivatives which are used as photochemical caging groups. Peters <i>et al.</i> (2009) report that upon photolysis, the nitrobenzyl group rearranges to the α-hydroxy-substituted nitrosophenyl group. The nitroso group then undergoes an additional reaction with the N-terminal amide group to generate the cyclic azo product. Subsequent hydrolysis of the activated carbonyl group results in the terminal cinnoline and carboxylate products (Peters <i>et al.</i>, 2009).

<p class="figure subtitle"><b>Figure 1b: Animation of the proposed mechanism of photocleavage reaction by Peters <i>et al.</i>, 2009.</b> The amino acids (blue) are linked to each other (black). The 2-NPA (purple) converts to its cinnoline form (cyclization) when irradiated with UV-light.

<p class="figure subtitle"><b>Figure 2:</b> Structure of 2-Nitrophenylalanine.

                     We use a recombinant fusion protein with streptavidin as binding unit and a medium gly-gly-ser-linker with 2-NPA connecting the streptavidin with a target protein. As a proof of concept, we used GFP as target protein because of its easy optical detection.

                     <br>We hope that the fusion protein in unfiltered cell lysate will bind strong and specifically to the <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Hardware">purification column</a> 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 485&nbsp;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 367&nbsp;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.  

<p class="figure subtitle"><b>Figure 4:</b> Three perspectives of the photoproduct of the tetrameric wild type <i>Aequorea victoria</i> green fluorescent protein (GFP) from rcsb.org.

<p class="figure subtitle"><b>Figure 5:</b> Seven versions of fluorescent proteins that emit light in different colors when exited with a specific wave length [http://pdb101.rcsb.org/motm/174].

                     Streptavidin is a tetrameric protein with a molecular weight of 15 kDa for each of the four subunits. It was isolated from the actinobacterium <i>Streptomyces avidinii</i> and is homologous to avidin. Both proteins can bind up to four molecules of biotin and their derivatives with high affinity resulting in the high dissociation constant Kd = 10-15 M. This leads to its widespread use in diagnostic assays and protein tags that require formation of an irreversible and specific linkage between biological macromolecules (Lichty <i>et al.</i>, 2005).  

<p class="figure subtitle"><b>Figure 6:</b> Three perspectives of the structure of a wild type streptavidin tetramer complex with biotin from rcsb.org.  

<p class="figure subtitle"><b>Figure 7:</b> Fusion protein of EGFP and Cytochrome b562 from rcsb.org.  

                     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 <i>et al.</i>., 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 designed depending on the desired characteristics. They are mainly classified in three different groups: flexible linkers (Figure 8 A), rigid linkers (Figure 8 B), and cleavable linkers (Figure 8 C). Furthermore they can be classified as small (approximately five amino acids), medium (approximately ten) or large linkers (approximately 20 to 28 amino acids) (Weber  <i>et al.</i>, 1989).

<p class="figure subtitle"><b>Figure 8:</b> Three groups of protein linkers. A: flexible, B: rigid, C: cleavable.  

<b>Xiaoying Chen, Jennica Zaro, and Wei-Chiang Shen</b> (2013). Fusion Protein Linkers: Property, Design and Functionality. Adv Drug Deliv Rev. <b>65(10)</b>: 1357–1369.<br><br>

<b>Lichty, J.J., Malecki, J.L., Agnew, H.D., Michelson-horowitz, D.J., and Tan, S.</b> (2005). Comparison of affinity tags for protein purification. <b>41</b>: 98–105.