I have expressed the kinase domain of my protein in E.coli fused with GST tag, and have purified it. But it seemed to have no activity in vitro. My protein is about 40KD, does the 26KD GST tag affect the conformation of my protein?
Tamjeed Saleh
GST in general is a good tag to enhance solubility of target protein. If you want to check whether the GST tag effects your kinase activity you can cleave the tag and test activity. You can also check to see if there are publications on successful expression of your kinase. But in general if you have a long enough linker between your GST and your KD, it should be ok. You may also want to check whether your kinase expressed in Ecoli is active (does it have the required phosphorylation, cofactors etc)
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