Debate

[Tom Anderson]

On Fri, 29 Feb 2008, DK wrote:

> In article <d9Txj.15694$097.6361 from newsfe21.lga>, aawara from pontiff-playground.org wrote:
> >In <sBSxj.3$DO4.0 from newsfe02.lga>,
> > DK <dk from no.email.thankstospam.net> wrote:
> >
> >>>There is a relatively new alternative to GFP for fluorescent tagging of
> >>>proteins in live cells in the shape of the FlAsH/ReAsH system:
> >>>
> >>>http://www.invitrogen.com/content.cfm?pageid=11922
> >>>
> >>>Basically, you put a magic tag on your protein,
> >>
> >> The magic tag with four exposed Cys is a subject to many
> >> crosslinking artifacts. Probably not a problem for most
> >> cytoplasmic proteins (reducing environment) but must
> >> be a huge problem for anything that goes through secretion
> >> pathway.
> >
> >Any thoughts on the SNAP tag?
> >http://www.covalys.com/products/snap-cell-labeling-in-cells.html
>
> Interesting! I did not know about it, thanks.
>
> Since the company does not seem to provide any concrete
> details, I had to download their plasmid sequence to BLAST
> and find out what "SNAP tag" is.
>
> Human 6-O-methylguanine DNA methyltransferase.
> Only ~ 20K, good, no tight dimers in the crystal, termini are
> not involved in the active site, so presumably works on both
> N- and -C end. The question what a ligand is and how
> tightly it binds seems to be answered by PDB code 1YFH :
>
> "Involved in the cellular defense against the biological effects of
> o6-methylguanine (o6-meg) in dna. This is a suicide reaction:
> the enzyme is irreversibly inactivated. Repairs alkylated
> guanine in dna by stoichiometrically transferring the alkyl
> group at the o-6 position to a cysteine residue in the enzyme."
>
> E.g., the binding is irreversible.
>
> All in all, seems to be appealing. I am contemplating some
> in vitro FRET and it maybe just what doctor ordered.

So what advantage over GFP and friends? 20 kDa is the same ballpark as
GFP, and we know that GFP has good monomericity and fusability. I guess
with SNAP you can use a chemical fluorophore, which can be much brighter
and more stable. And of course, you get to choose the colour *after*
you've expressed the protein, which is nice!

Is there a solution for two-colour SNAP? You'd need another modified
version of the enzyme that only recognised some other form of substituted
nucleotide, i suppose.

tom

-- 
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264   (f) +44 (20) 76797805   (e) thomas.anderson from ucl.ac.uk