Expression Problem - fusion protein

[Mega [Andreas Stuermer]]

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

[Mega [Andreas Stuermer]]

Just noticed, it would also be possible to change the second amino acid from isoleucine to valin/alanine/Glutamic acid/Glycine/aspartic acid  to create the same restriction site.


Just had a look at valine, and it looks quite the same :D

[Cathal Garvey]

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".

[Richard Yu]

Yep, the second residue determines cleavage efficiency, and then also influences protein lifespan (N-end rule).

According to http://www.mcponline.org/content/5/12/2336.full.pdf, in Ecoli, 

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.  

N end rule in  e coli, from Varshavsky's 1991 Science paper:

Amino-terminal arginine, lysine, leucine, phenylalanine, tyrosine, and tryptophan confer 2-minute half-lives on a test protein; the other amino-terminal residues confer greater than 10-hour half-lives on the same protein. 

If you had a choice, I'd suggest valine.

Cheers,

Rich

[Nathan McCorkle]

On Mon, Oct 7, 2013 at 1:34 PM, Richard Yu <r...@radiantgenomics.com> wrote:
> Yep, the second residue determines cleavage efficiency, and then also
> influences protein lifespan (N-end rule).

Huh, that's a new thing to look up I hadn't heard of.

> N end rule in e coli, from Varshavsky's 1991 Science paper:
> Amino-terminal arginine, lysine, leucine, phenylalanine, tyrosine, and
> tryptophan confer 2-minute half-lives on a test protein; the other
> amino-terminal residues confer greater than 10-hour half-lives on the same
> protein.

Very very interesting, in all the cell and molbio classes I've had, I
don't remember hearing about this.

[Ravasz]

I didn't know about this half life thing either. But I did add a few AA-s to ends of various proteins for cloning purposes, and all of them worked so far. So I would say adding just a few extra amino acids to the N term should still work most of the time, as long as it starts with Met. :)

[Mega [Andreas Stuermer]]

Ok thanks!

Especially your experience was helpful, ravasz.

However I could circumvent the problem by choosing a different similar UTR of the literature :D
So I have gaurantee that it works.

I guess wiuth one protein it's ok, but if you have a multi-enzyme complex, it's another story...

[Mega [Andreas Stuermer]]

I guess if you have a multi-enzyme complex, some weird interaction with the N-Terminus are more likely than in single enzymes...

[Cathal Garvey]

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]" <masters...@gmail.com> wrote: