Disulfide bond formation is an important protein modification that is mostly found in secreted and membrane proteins often supporting their structure and stability. In eukaryotes, the formation of disulfide bonds is driven by the oxidative environment of the endoplasmic reticulum (ER), and thus is separated from the reducing environment in the cytosol. We are trying to mimic this separation of reducing and oxidizing conditions using our bilayer reaction format.
In a bilayer reaction, protein synthesis mostly happens in the lower layer containing the wheat germ extract providing the ribosomes and supporting factors. The translation reaction in the lower layer is fed by the reaction buffer in the upper layer. Fresh energy and amino acids are supplied to the translation reaction by diffusion between both layers mixing over time. As shown in the figure below, we used this approach to work with different buffers in both layers yielding in an oxidative environment at the end of the reaction to better stabilize proteins having disulfide bonds.
Disulfide bond formation is further supported by different enzymes in the ER. In our products, we are using a human endoplasmic reticulum oxidoreductase (Ero1α) and a human protein disulfide isomerase (PDI), which are enzymes catalyzing the formation and breakage of disulfide bonds thus supporting the correct arrangement of disulfide bonds in their target proteins.
Refer to the application notes on the preparation of an active tissue plasminogen activator (tPA) protease domain and IgG Antibody Fab fragment to learn more on how these reaction conditions can enable the preparation of functional proteins having disulfide bonds.
Application Notes
Synthesis of tissue plasminogen activator (tPA) protease domain with retained activity
Synthesis of IgG Antibody Fab fragment
Products
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Disulfide Bond PLUS Expression KitEntry kit, designed for ease use to test reaction conditions for disulfide bond formation. This product supports 8 transcription and translation reactions working on 230 µl scale. |
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Disulfide Bond Enhancer Enzyme SetThis product can be used it in combination with the WEPRO7240H Core Kit and provides all reagents to modify bilayer reaction for the synthesis of proteins having disulfide bonds. The Core Kit format supports working with the bilayer format at different reaction scales. |
* We recommend not to use these products if the protein should later be purified by means of a GST-tag. Disulfide bonds are not stable when working with glutathione containing buffers to elute the protein.
Inquiry
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