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 signals, nor am I aware of any other groups that have tried other signal sequences in Pichia with this family of proteins. We have been extracting proteins from the pellets by a quick alkaline extraction method, variations of which have been published for S. cerevisiae and S. pombe; to judge from our Coomassie stained gels, it is pretty efficient for the Pichia as well. I'd never before thought of checking the homologous residues in the other family members that have been produced in Pichia. The residues at these positions by sequence alignment are D, P, G, and S. However, when I do a structure-based alignment with the one other family member that has been crystallized, I see that structural conservation at these sites is poor; they are on loops that look completely dissimilar between the proteins, so I don't think I can read much into the substitutions. Thanks for your help.
Evette Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab) -----Original Message----- From: Stephen Weeks [mailto:[EMAIL PROTECTED] Sent: Wednesday, March 05, 2008 10:43 AM To: CCP4BB@JISCMAIL.AC.UK; Radisky, Evette S., Ph.D. Subject: Re: Removal of glycosylation sites in Picha expression construct 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
