Peptide/Amino Fusion by Chemistry

[Cathal Garvey]

Hello all,
I'm trying to puzzle out the practicality of a standardised method for
peptide fusion in vitro. Basically, to take two peptides or amino backbones,
and fuse one to the other chemically somehow. Assuming both could be
arranged to have uncomplicated N/C terminal ends, how does one go about
bonding them together? Is it reliable? Easy?

Many thanks,
Cathal Garvey
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[Nick Theodorakis-2]

On Oct 15, 10:32 am, Cathal Garvey <cathalgar...@...> wrote:
> Hello all,
> I'm trying to puzzle out the practicality of a standardised method for
> peptide fusion in vitro. Basically, to take two peptides or amino backbones,
> and fuse one to the other chemically somehow. Assuming both could be
> arranged to have uncomplicated N/C terminal ends, how does one go about
> bonding them together? Is it reliable? Easy?
>
> Many thanks,
> Cathal Garvey

There are several ways that are used to make synthetic peptides; you
can review then at the wikipedia article here:

<http://en.wikipedia.org/wiki/Peptide_synthesis>

Is this similar to what you had in mind, or were you thinking about
something different?

Nick

--
Nick Theodorakis
nick_theodorakis@...
contact form:
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[WS-4]

Hi Cathal,

if there are no reactive side chains and you "just" want to join the
carboxy terminus to the amino terminus (i.e. generate a cyclic
peptide), then it might be easy with EDC and NHS in aqueous / buffered
solution. Don't use amine and carboxylate based buffers of course
(e.g. tris, acetate etc), as they will quench your reagents. Phosphate
or borate should work. After the reaction, cleanup may be done with
gel filtration, dialysis or an organic / aqueous extraction, depending
on the size of the peptide / protein. Detailed adaptable protocols
should be available from PubMed and / or Google by searching for "EDC"
and "NHS". People use this approach usually for linking haptens to
carrier proteins like BSA, KLH or to label antibodies and proteins
with dye molecules.

However, if you plan to fuse two peptides (both with an N and a C
terminus) and / or have reactive side groups (lysine, glutamate,
aspartate), then best ask a dedicated lab for assistance. You'll need
a lot of protection (and subsequently deprotection) chemistry.

Good luck!

HTH

Wo
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[Cathal Garvey]

Thanks for the detailed replies!
After doing some reading yesterday on the matter, it seems far less simple
than I'd thought it would be.

To refine what I was asking, what I'm looking at doing is using a
synthesised strand of Peptide Nucleic Acid, which would have an N and C
terminus and would probably lack protection (aside from whatever blocking
groups were used to build the chain). To that I'd aim to fuse a protein
produced in the lab by e.coli, which I think is expected to have natural
protection or blocking groups on either terminus (?).

I have read a little about inteins, which seem to be a clever way of having
protective groups cut themselves off. If they work well enough to generate a
large quantity of unprotected protein, it should mean both of my fragments
(PNA and protein) are unprotected at the relevant termini.

So, for unprotected termini and straightforward N-C fusion, what would you
recommend as an in-lab fusion reaction? Please note that I'm a molecular
biologist by trade, rather than an organic chemist.
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[David-Paul Minde]

Hi Cathal,
I think the straightforward way is using sortase a reaction for your
fusions:
one c-terminus has LPXTGXX motif and is fused to another n-terminus via
sortase a enzyme.
The beauty of the system is specificity, short recognition motifs (no add.
cloning required ;) virtually no influence on expression of target proteins,
compatibility with diverse coupling chemistries inlcuding PNAs according to
an exploding literature in recent years.
Sortase a can be easily expressed in bacteria.
Introduction of "LPXTGGG" linker motif may be the only major disadvantage.
Intein is also fine in some cases and more puristic chemistry. However,
extreme variability of efficiency generally "kills" many intein fusion
attempts.
cheers,
David

2009/10/16 Cathal Garvey <cathalgarvey@...>

> Thanks for the detailed replies!
> After doing some reading yesterday on the matter, it seems far less simple
> than I'd thought it would be.
>
> To refine what I was asking, what I'm looking at doing is using a
> synthesised strand of Peptide Nucleic Acid, which would have an N and C
> terminus and would probably lack protection (aside from whatever blocking
> groups were used to build the chain). To that I'd aim to fuse a protein
> produced in the lab by e.coli, which I think is expected to have natural
> protection or blocking groups on either terminus (?).
>
> I have read a little about inteins, which seem to be a clever way of having
> protective groups cut themselves off. If they work well enough to generate
> a
> large quantity of unprotected protein, it should mean both of my fragments
> (PNA and protein) are unprotected at the relevant termini.
>
> So, for unprotected termini and straightforward N-C fusion, what would you
> recommend as an in-lab fusion reaction? Please note that I'm a molecular
> biologist by trade, rather than an organic chemist.
> _______________________________________________
> Methods mailing list
> Methods@...
> http://www.bio.net/biomail/listinfo/methods
>

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[StewJW]

A good start would be to drop the fusion terminology and define where
you want your covalent linkage formed. If your going to go down the
synthetic route I'd seek advice from a peptide chemist. If you just
want to perfom a simple cross-linking reaction Pierce has one the most
comprehensive range of reagents around. They also have a handbook
which offers advice:

http://www.piercenet.com/Objects/View.cfm?Type=Page&ID=FE7F690D-58AE-4342-AE85-BA94DCA642F8


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[Dr Engelbert Buxbaum]

Am 16.10.2009, 04:14 Uhr, schrieb Cathal Garvey <cathalgarvey@...>:


> To refine what I was asking, what I'm looking at doing is using a
> synthesised strand of Peptide Nucleic Acid, which would have an N and C
> terminus and would probably lack protection (aside from whatever blocking
> groups were used to build the chain). To that I'd aim to fuse a protein
> produced in the lab by e.coli, which I think is expected to have natural
> protection or blocking groups on either terminus (?).

It would probably be a lot easier to make a new DNA construct and express  
that. The chemistry involved in your fusion reaction might be feasible, if  
complicated, in peptides. However, it would almost certainly destroy the  
secondary and tertiary structure of a protein.

If you could live with linking peptide (e.g. a hapten) and protein (as  
carrier) at some reactive residue, rather then a C-to-N linkage, that  
would be a lot simpler, and there are a lot of tried procedures out there  
(think HRP-antibody conjugates).
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