Debate

[Tom Anderson]

On Sat, 1 Mar 2008, Aawara Chowdhury wrote:

> In <u%Uxj.22$TP4.13 from newsfe07.lga>,
>  DK <dk from no.email.thankstospam.net> wrote:
>
> > One thing I am confused about: since its substrate is
> > in the nucleus, you'd expect it to be targeted to nucleus?
> > And yet, the SNAP is a full lenght human cDNA and the
> > company provides a special SNAP plasmid that targets
> > the tag to nucleus. How come? Don't all enzymes that
> > work on chromosomes tend to localize to nucleus?
>
> Those are good observations and questions.  I haven't used the tag,
> but here's the information that I had received earlier.  The SNAP tag
> is a modified version of 6-O-methylguanine DNA methyltransferase, that
> only recognizes para-substituted benzyl guanines.  This substrate is
> not recognized by the parental enzyme (and it is claimed, other cellular
> proteins).
>
> The substrate (para-substituted benzyl guanine) is membrane permeable,
> so it gets into cells very easily, and by the reaction you described,
> modifies the SNAP tag with the para-substituent (which is typically
> a fluorophore).
>
> Since nucleotides/nucleosides are both cytoplasmic and nuclear,
> presumably the substrate goes to where the SNAP-tagged protein is.
>
> My concern is the same as yours.  Does the tagged protein get dragged
> to the nucleus?

>From their document 'Wild-type AGT does not give any back-ground when
labeling SNAP-tag fusion proteins in mammalian cells' [1]:

"Wild-type AGT contains a DNA binding domain, resulting in a predominant
nuclear localization, and reacts preferentially with DNA-bound alkyl
guanine. Even though it is derived from human AGT, SNAP-tag is highly
modified and optimized for use as a self-labeling tag. DNA-binding is
abolished, leading to uniform distribution of SNAP-tag throughout the
cytosol and the nucleus, and it is optimized to react rapidly with its
free benzyl guanine and pyrimidyl guanine substrates."

They say they've deleted the DNA-binding domain, so no nuclear
localisation. DK, are you sure it's a full-length cDNA?

tom

[1] http://www.covalys.com/fileadmin/documents/SNAP_cell_no_wtAGT_labeling_visible.pdf

-- 
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