Could be a stupid question but - (Dec/08/2008 )

Does anyone know if BL21(DE3) are capable to phosphorylate proteins? It's strange i know but justified a lot of things

Thanks in advance

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E.coli do not contain detectable tryrosine kinase, that's why a derivative bacteria TKB1 cells (containing inducible receptor tyrosine kinase) have been used to identify RTK interacting proteins. But I think E.coli have many other kinases (such as NDP kinase, aspartate-, threonine-, methionine-, lysine-kinase etc). What does that mean in relation to your question? I don't know. Let somebody else jump in.

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Hi,

Does that mean your recombinant proteins are already phosphorylated?
Because we've experienced in our lab that some recombinant kinases are already phosphorylated when expressed in E.coli BL21. Not sure if that's because of E.coli kinases or because of autophosphorylation. We're thinking it might be the latter.
You can maybe try a Western with for example anti-phosphor-Tyrosin AB (depending on what phosphorylation you're expecting) to check if they already are phosphorylated...

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I suspected that protein is phosphorylated and that is a problem to my phosphorylation assays. I'm try figure it out if this really happen but has been complicated to get unequivocally results. I will have to try a different approach...

Thanks for replies

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