Re: [DIYbio] Re: Expression Problem - fusion protein

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I don't see why; there's only one N-terminus, and it should obey all
the usual rules.

Indeed, if you're fusing proteins, changing the N-terminal peptide is a
relatively minor operation. Fusing proteins is one of those things that
either works great or not at all, in my experience. I recently fused
two proteins that work great individually, and got high expression of
the fusion protein..but one domain simply didn't work anymore. I'm now
awaiting a re-synthesis of the same combination, but with the
non-functioning domain at the C-term instead of N-term, to see if that
works any better. In your case you may encounter something similar.

Also, when fusing proteins it's generally advised to put in a "spacer"
of about 6 amino acids; preferably simple, soluble and (IMO) not
proline. These help insulate the N-term of one fused protein from the
C-term of the other, and helps prevent the second domain from being
pressed against the prior domain by a rigid C-terminus, preventing
folding. All of which could be black magic, who knows.

On Tue, 8 Oct 2013 04:42:45 -0700 (PDT)
"Mega [Andreas Stuermer]" <masterstorm123@gmail.com> wrote:

> I guess if you have a multi-enzyme complex, some weird interaction
> with the N-Terminus are more likely than in single enzymes...
>
>
>
>
>
> On Monday, October 7, 2013 4:47:03 PM UTC+2, Mega [Andreas Stuermer]
> 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
> >
>