Monomeric forms of fluorescent proteins like GFP may aggregate when they are fused to other proteins which are expressed under high levels from strong promoters. One of the solutions of this problem is to use genetically modified fluorescent proteins that are unable to aggregate. Does anyone know which amino acid substitutions in GFP or other fluorescent proteins prevent their aggregation?
Mutations A206K, L221K, and F223R were found to essentially eliminate self-association of GFP/YFP/CFP, as reported in D.A. Zacharias et al. (2002) Science 296:913-916. A good overview of these and other studies to improve fluorescent tools for live cell imaging is found in a 2005 review by Roger Tsien (R.Y. Tsien (2005) FEBS Lett. 579:927-932.
Check out tetracystein tag. I switched from GFP fusion because it cause loss of fluorescence of the fused protein. I now use TC tag. Check out the paper by Hoffmann et al., 2010 Nature Protocol. It seems it's gaining popularity.
How is to use mChrry tag (Clonetech)? It knows that exist by monomeric form, natively. I am changing to mCherry because of GFP's dimerization.
If you use the A206K mutant, it should stay monomeric even in the cell cytoplasm. Otherwise SNAP-tag is an elegant solution to replace GFP fusion.
Mutations A206K, L221K, and F223R were found to essentially eliminate self-association of GFP/YFP/CFP, as reported in D.A. Zacharias et al. (2002) Science 296:913-916. A good overview of these and other studies to improve fluorescent tools for live cell imaging is found in a 2005 review by Roger Tsien (R.Y. Tsien (2005) FEBS Lett. 579:927-932.
Take care about the mutation L221K on GFP aequoria victoria derivatives. see Kim et al, Molec Biosystem 2009
see also the role of the A206K in the CFP dimerisation as discussed in Espagne et al (2011) Biochemistry 50, 437
Article The use of Cyan Fluorescent Protein variants with a distinct...
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