>Date: 17 Mar 1995 15:19:14 -0000
>Sender: lpddist at mserv1.dl.ac.uk>Message-ID: <3kc99i$nvt at mserv1.dl.ac.uk>
>Original-To: glycosci at dl.ac.uk>>I have produced a recombinant protein in the yeast Pichia pastoris that is
>partially glycosylated with O-liked sugar (there are no N-linked sites in the
>protein). I wish to seperate the glycosylated form the unglycosylated protein.
>I have tried using ConA-Sepharose (Pharmacia) and Glycine-Max (Sigma) without
>success (neither seemed to bind the glycoprotein). The ConA came with
>instructions, but I have not been able to dig out any protocol for using the
>Glucine-Max and Sigma were not forthcoming.
Going by the 'rules', Con A would not bind O-linked mannose. (See
Kobata and Yamashita in Glycobiology A Practical Approach, p. 110 -
they refer to a previous Kobata paper and state that ConA 'interacts
with sugar chains which have two non-substituted or C-2 substituted
alpha-mannopyranosyl residues in one molecule') That said, they
probably did not study O-linked mannose. According to another article
in the same book (Berlin and Hanover, p. 343). Soybean agglutinin
(Glycine max) would bind D-GalNAc (but there's no more detail than
that). I don't know whether Clinton Ballou or Widmar Tanner (who have
looked at yeast O-mannosylation) have examined any lectin binding to
O-mannose.
The question is do you really want this protein glycosylated or not?
The only problem with yeast mutants of this pathway is that (if I
remember the work of Peter Orlean on Dol-P-Man synthetase mutants)
the O-mannosylation is required for viability. Also Tanner's work
appears to indicate that there are two transferases for the first
step in O-mannosylation.
A novel way round your problem may be to investigate the specificity
of mannose binding protein (animal origin) and see if a recombinant
form of MBP could bind your enzyme!
---------------------------------------------------------------
Iain Wilson Institut fuer Chemie
Tel: 43-1-47654-6065 Universitaet fuer Bodenkultur
Fax: 43-1-310-5176 Gregor-Mendel-Strasse 33
E-mail: wilson at edv1.boku.ac.at A-1180, WIEN, Austria
http://www-bprc.mps.ohio-state.edu/cgi-bin/hpp/iain_wilson.html
