Hi Evette,
Have you tried to mutate one glycosylation site at the time and check
for expression? Often glycosylation is necessary for secretion -that's
the case with my protein. Also these sites tend to have a defined
glycosylation pattern which eliminates the need of deglycosylation.
You
Dear Evette,
it is quite common that mutation of glycosylation sites completely kills
expression. One rationale is that the glycans cover hydrophobic patches
on the surface of the protein, and when exposed, the protein won't fold
properly anymore.
Pichia is a pain because the glycans it produces
Evette,
As the glycosylation seems to be critical for production of your
protein in P. pastoris I would imagine it would be the same in both
insect and mammalian cells. As I'm sure you've invested plenty of time
getting this expression system to work it would seem a waste not to try
Thanks to everyone for the great suggestions so far. To clarify and
answer a few questions, with the mutated construct we get no protein
either secreted or in the pellet. The protein in question is about 20
Kda; with glycosylation at both sites it is around 29 kDa (both in
mammalian cells and
Evette,
This is quite intriguing. At the outset I want to say that just because
glycoslyation is not essential for function it still may be absolutely
necessary for correct trafficking.
Obviously in your case the mutant has been made successfully in CHO
cells, assuming the mutant
2008 08:38:46 -0600
From: [EMAIL PROTECTED]
Subject: Re: [ccp4bb] Removal of glycosylation sites in Picha expression
construct
To: CCP4BB@JISCMAIL.AC.UK
Thanks to everyone for the great suggestions so far. To clarify and
answer a few questions, with the mutated construct we get
Stephen,
We confirmed by PCR that our gene was chromosomally integrated in the
many Pichia colonies that we screened for expression, but we did not
directly assess for mRNA transcripts. We are indeed using the alpha
mating factor secretion signal and have not tested any alternative
secretion
Radisky, Evette S., Ph.D. wrote:
The residues at these positions by sequence alignment are D,
P, G, and S.
Don't Perturb Glycosylation Sites. Sorry, I couldn't resist.
--
Paul Paukstelis, Ph.D.
Research Associate
Institute for Cellular and Molecular Biology
The University of Texas at Austin
-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Radisky, Evette S., Ph.D.
Sent: Wednesday, March 05, 2008 9:39 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Removal of glycosylation sites in Picha expression
construct
Thanks to everyone for the great suggestions so far
Dear all,
Our lab is new to working with Pichia pastoris, also new to working with
glycosylated proteins. We have a construct for a secreted protein that
expresses pretty well in Picha, but upon mutation of the 2 N-linked
glycosylation sites to Ala, we get no expression at all, nada. The
This is not entirely uncommon. Did you try removing just one of the two
sites (sometimes it helps) - combined with enzymatic deglycosylation this
may give you good enough protein to work with.
If you try other hosts, I would definitely consider Schisosaccharomyces
pombe - its glycosylation
folding in your
case.Holly Jing
Date: Tue, 4 Mar 2008 16:54:00 -0500 From: [EMAIL PROTECTED] Subject: Re:
[ccp4bb] Removal of glycosylation sites in Picha expression construct To:
CCP4BB@JISCMAIL.AC.UK This is not entirely uncommon. Did you try removing
just one of the two sites (sometimes
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