troubleshooting GST fusion protein

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roudi
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troubleshooting GST fusion protein

GST fusion protein expression in Ecoli needs a lot of optimization. I have had problems in solubilization process. I use triton X-100 after the cell harvest. Is this detergent a good choice?

Soudabeh
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roudi wrote:GST fusion

roudi wrote:

GST fusion protein expression in Ecoli needs a lot of optimization. I have had problems in solubilization process. I use triton X-100 after the cell harvest. Is this detergent a good choice?

To enhance solubilization is recommended to use a combination of TritonX-100 and sarkosyl. This method consists of solubilizing the protein in the presence of sarkosyl and EDTA prior to sonication, followed by clarifying the lysate and adding Triton-X prior to purification over Glutathione sepharose. The ratio of sarkosyl : TritonX-100 is empirically determined in order to optimize solubilization of protein and binding to Glutathione Sepharose.

Paul Weinreb
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Do you know whether the

Do you know whether the protein is expressed in soluble form to begin with? Many proteins are expressed in insoluble form in inclusion bodies, and your best bet is to try and avoid this to begin with. One simple solution that has worked for us is to lower the expression temperature from 37°C to 30°C or even 25°C (need to let the cells grow longer, obviously). Also reportedly adding magnesium to the cells works. Finally, there are strains of E coli with chaperones built into them that supposedly help expression (e.g. "Origami") but I don't have any personal experience with them.

Paul Weinreb
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Minor point, but you may want

Minor point, but you may want to move this discussion to a different board (e.g. Biochemistry) since a GST fusion is not an Antibody (e.g. Fc) fusion protein.