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ProteOlux ®

Proteolux P

As shown previously the Proteolux system offers a light-inducible specific mechanism to degrade tagged protein. This system may be applied to any protein expressed in the cytoplasm of the host cell. For the original system a homologue recombination step is necessary in order to fuse the PIF-DAS tag to the target protein in the host cell or organism. This step requires additional time in order to prepare the experiment and might further change the normal protein physiology, which should be observed, due to the creation of a fusion protein. It is known, that the function of a fusion protein can be different of the wild-type protein due to different stability of the mRNA after the homologue recombination leading to a different translation rate or the expressed protein does not fold correctly and forms aggregates in the cytoplasm. These drawbacks of the original system are solved in Proteolux’s P (Plus and Pro) system.

The Proteolux Plus and Pro system allows the observation of the native target protein functions without long experiment preparation and interference with the cell metabolism. The developed strategy is to use a specifically engineered intrabody against the target protein. The binding of the intrabody to the protein can lead to the direct inhibition of the protein function. In the case, that the protein is not directly inhibited and to prevent re-establishment of the protein function the intrabody-protein complex is targeted to the proteasome. The binding of the intrabody to the target protein has to be regulated to avoid constant degradation of the target protein. Therefore, Proteolux offers the Plus and Pro system of different complexity, according to the experimenter demand.

Intrabodies are antibody derived proteins that bind to an intracellular protein within the cell. The normal antibody (figure X) has to be modified due to the reducing environment within the cytoplasm that prevents disulfide bridge formation. Therefore, single-chain antibodies, which are fusion proteins of the variable region of the light chain (VL) and the heavy chain (VH) connected by a short linker, are often expressed.

Proteolux Plus ®

Intrabody expression regulation by Proteolux’s light inducible degradation system.

It has been shown (A-P.Sibler & al. 2005) that the C-terminus fusion of a PEST sequence (a protein region rich in proline, glutamic acid, serine and threonine) to an intrabody is a key element for binding to the control region of the 26S proteasomes (Zhang M. & al. 2003). This modification leads to a shortened half-life of the intrabody which is transferred to the bound target protein as it is degraded together with the intrabody. These PEST-tagged intrabodies of sufficient affinity and solubility are powerful tools to shorten/decrease the half-life of the target protein.

For the controlled regulation of the target protein, the destabilized intrabody is controlled by a strong eukaryotic promoter, which is repressed by a strong repressor. The used repressor is tagged with the PIF-DAS tag construct and is constitutively expressed. Consequently the expression of the destabilized intrabody will be repressed until the repressor is degraded by a red light pulse. Once the intrabody containing the PEST sequence is expressed, it binds to the target protein and targets the bound protein to the proteasome. The intrabody is expressed continuously until the degradation of the repressor degradation is stopped by a far-red light pulse.

The functioning of the Proteolux Plus