4 replies to this topic

[Wek]

I have read (cant remember where) that one can fuse a phosphate molecule to a protein permanently and therefore constitutively activate it. I have searched P-O and pubmed but haven't found anything. Does anyone know where I can find more info about this technique?

Thanks

[DRT]

Would making sure there are no phosphatases around do the trick? Or are you hoping to have the protein working in vivo?

[Wek]

I'm hoping it would work in vivo.

[mdfenko]

we used atp-gamma-s as the source of phosphate during phosphorylation. the sulfur makes a phosphatase resistant link.

[allynspear]

If it is possible in your system, you can also mutagenize the phosphorylated Serine or Threonine to an Aspartate or Glutamate.  The presence of the negatively charged group can partially simulate phosphorylation and can give rise to a permanently active (or inactive) expressed protein.  This may not work for all proteins, but it has been used successfully in many published studies.

Best of Luck.