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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 releasing an active form, or a combination with other methods as demonstrated in our light-induced elution. | 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 releasing an active form, or a combination with other methods as demonstrated in our light-induced elution. | ||
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The ncAA 2-NPA is able to photochemically cleave the polypeptide backbone by an cinnoline forming reaction based on the photochemistry of (2-nitrophenyl)ethane derivatives which are used as photochemical caging groups. Peters et al. (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 affords the terminal cinnoline and carboxylate products [3]. | The ncAA 2-NPA is able to photochemically cleave the polypeptide backbone by an cinnoline forming reaction based on the photochemistry of (2-nitrophenyl)ethane derivatives which are used as photochemical caging groups. Peters et al. (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 affords the terminal cinnoline and carboxylate products [3]. | ||
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<p class="figure subtitle"><b>Figure 1: Proposed mechanism of photocleavage reaction by Peters et al [3].</b> | <p class="figure subtitle"><b>Figure 1: Proposed mechanism of photocleavage reaction by Peters et al [3].</b> | ||
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Revision as of 01:35, 27 August 2017
Photolysis
Short summary
Photolysis of peptide chains
Explanation of the ncAA
Figure 1: Proposed mechanism of photocleavage reaction by Peters et al [3].
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
Theoretical basis
Green fluorescent protein: GFP
Figure 1: Three perspectives of the photoproduct of the tetrameric wild type Aequorea victoria green fluorescent protein from rcsb.org.
Figure 2: Three perspectives of the photoproduct of the tetrameric wild type Aequorea victoria green fluorescent protein from rcsb.org.
Streptavidin
Figure 2: Three perspectives of the structure of a wild type streptavidin tetramer in complex with biotin from rcsb.org.
Fusion Proteins
Figure 3: Fusion protein of EGFP and Cytochrome b562 by rcsb.org.
Figure 4: Three groups of protein linkers. Left: flexible, middle: rigid, right: cleavable [6].
Light-induced elution
Figure 5: 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.