Methionine aminopeptidase (MAP) is a ubiquitous, essen- tial enzyme involved in protein N-terminal methionine ex- cision. According to the generally accepted cleavage rules for MAP, this enzyme cleaves all proteins with small side chains on the residue in the second position (P1), but many exceptions are known. The substrate specificity of Escherichia coli MAP1 was studied in vitro with a large (>120) coherent array of peptides mimicking the natural substrates and kinetically analyzed in detail. Peptides with Val or Thr at P1 were much less efficiently cleaved than those with Ala, Cys, Gly, Pro, or Ser in this position. Certain residues at P2, P3, and P4 strongly slowed the reaction, and some proteins with Val and Thr at P1 could not undergo Met cleavage.
If you had a choice, I'd suggest valine.
There can be *no* guarantees in biology, of anything.
However, adding two amino acids to the N-terminus of your protein is
unlikely to destroy everything.
Do bear in mind; if the methionine will be excised from the protein,
which itself depends heavily on the second amino, then the second amino
will be the N-terminus of your protein; and the N-terminal amino has a
strong-ish impact on the long-term stability of the protein, with some
aminos (like Valine) being highly stable and others being highly
unstable.
I can't recall offhand but I think likelihood-of-M-excision is higher
if the first few aminos are..simple unbranched/uncharged? Someone else
wanna back me up or correct me here? So Glycine might encourage
methionine processing, and Glycine is probably unstable-ish.
As usual then, the answer is "probably not a big deal but there's all
this random stuff that might go wrong and in the end it might explode
or work amazingly".
On Mon, 7 Oct 2013 07:47:03 -0700 (PDT)
"Mega [Andreas Stuermer]" <masters...@gmail.com> wrote:
> Hi everyone!
>
> For expression of a protein I would need to make it fit to the RBS of
> the vector.
>
> Is it ok, if I attach ATG-GGC (Methionine and Glycine) to the
> N-Terminus?
>
> So the fusion protein would be Met-Gly-Met-rest of Protein
>
> Is there a guarantee that the protein still works then?
>
> Best,
> Andreas
>
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