Peptide/Amino Fusion by Chemistry

David-Paul Minde via methods%40net.bio.net (by davidminde from gmail.com)
Fri Oct 16 11:06:35 EST 2009


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 from gmail.com>

> 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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