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production of phosphorylated proteins in E.coli host system

expression phosphorylation proteins E.coli

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#1 NaruBee

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Posted 05 August 2013 - 06:23 PM

Hi everyone,

 

I have no experience on kinase or phosphorylation but plan to do experiment on this field. 

 

I am planing to check the interaction of candidate phosphoproteins with my target protein.  So I would like to produce phosphorylated proteins in E.coli. If I know only what the protein is, the site of phosphorylation, is it possible to guess what kind of kinase is used for the phosphorylated protein? and I know the state of cell that express the phosphorylated proteins.

 

 If it is possible. Do you think which way is better for phosphorylation between co-expression both proteins in E.coli, or produce kinase proteins separately and perform in vitro phosphorylation between 2 proteins.  

 

Thank you in advance for suggestion.

 

-NKB-



#2 labtastic

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Posted 04 September 2013 - 12:52 PM

I think your best bet is to find a kinase that will phosphorylate your protein in vitro. Co-expression of a kinase with the phosphorylated protein might work, but I would worry about endogenous phosphatases creating homogeneity. 

 

A common alternative is to mutate the site of phosphorylation to an Asp or Glu. The negative charge on those acidic amino acids mimic the negative charge of a phospho group. This strategy has been shown to work quite well for some proteins, usually best as a phospho-serine mimic. 

 

There was a paper in Science about two years ago that reported site-directed incorporation of phosphoserine. I personally spent about 3 months attempting to replicate their results in any way possible with multiple different proteins, but was not successful. Last I checked no lab (except their own) has cited that work having actually produced phosphorylated protein of their own. If you come across this paper, my advice would be to stay away from that particular technology until improvements are made. When such improvements are made and a robust phosphoserine (or Tyr) incorporation technology is developed, it will be a game changer for protein chemistry no doubt.


Edited by labtastic, 04 September 2013 - 03:29 PM.






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