E-mail From NEB on Dec 9th
2015-01-13azim58 - E-mail From NEB on Dec 9th
Dear Kurt ,
Thank you for contacting NEB Technical Support. Your question and answer
are below.
Your question:
Oh actually it sounds like you may have exactly what I want! I want to
express an scFv (single chain Fragment variable) protein as a fusion with
the p3 M13 protein. An scFv gene is only about 750 bp and is like a mini
antibody containing only the heavy chain variable region, light chain
variable region, and a linker connecting the two. I think I can just
insert my scFv gene sequence instead of a sequence for a peptide if I am
not mistaken. Does your vector contain a SfiI site and NotI site that
would allow me to clone my peptide or scFv sequence into the vector?
Could I get a link to your system(s) applicable for this? Thanks for any
information you can offer me!
Our answer:
We have anecdotal evidence that large peptides may be displayed attached
to pIII but we generally say 50 amino acids is the max. We do know some
peptides/fragments display better than others so your 750 bp fragment is
worth a shot. Unfortunately, to keep the pIII leader sequence intact, we
did not design multiple restriction sites at the pIII insertion point.
You may be able engineer a couple others in there but we exclusively use
KpnI and EagI for cloning. The phage display vector is called M13KE and
the RF DNA is available in E8101S PhD Phage Display Cloning System,
http://www.neb.com/nebecomm/products/productE8101.asp
Regards,
Beth