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 transcript is present in Pichia I wonder if this
is an effect of fusing it to the alpha mating factor secretion signal. Have
you - or anyone else for that matter - tried alternative secretion signals ?
Sorry but I have two more questions for you. How do you lyse you cells to
see production of the protein in the pellet ? I found boiling Pichia in SDS
buffer pretty inefficient, do you use glass beads ? Secondly I’m curious as
to what residues are present when you align you protein to the related
non-natively glycosylated proteins (that where successfully expressed in
Pichia) specifically at the site of glycosylation.
Stephen
--
Stephen Weeks, Ph. D.
Drexel University College of Medicine
Department of Biochemistry and Molecular Biology
Room 10102 New College Building
245 N. 15th St.
Philadelphia, PA 19102
Phone: (+) 215-762-7316
Fax: (+) 215-762-4452
