[ccp4bb] Removal of glycosylation sites in Picha expression construct
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 nucleic acid sequence appears to be correct, i.e. we have not introduced any unintentional frame shifts, stop codons, or anything like that. Is this a common phenomenon? Are there any tricks to get the Pichia to do its thing? Any chance that alternative substitutions will work when Ala does not? Or are we better off (a) trying to deglycosylate enzymatically, or (b) trying a different expression host? All opinions and anecdotes welcome. Thanks! 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)
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
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 patterns are much simpler. Other likely candidates include regular bakers' yeast and Hansenula polymorpha. I would try both the native and the mutant proteins in those hosts. There are tricks one can play with Pichia to try and make it express the deglycosylated protein but it almost sounds like you've hit on something very essential since you're going from good expression down to no expression. Lastly, you could try insect cells :) Artem > > 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 > nucleic acid sequence appears to be correct, i.e. we have not introduced > any unintentional frame shifts, stop codons, or anything like that. Is > this a common phenomenon? Are there any tricks to get the Pichia to do > its thing? Any chance that alternative substitutions will work when Ala > does not? Or are we better off (a) trying to deglycosylate > enzymatically, or (b) trying a different expression host? All opinions > and anecdotes welcome. > > Thanks! > 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) > >
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
There are many cases in literature where the removal of N-linked glycosylations sites reduced the expression levels of secreted proteins. I would recommend removal of the N-linked glycans by enzymes (endo H, PNGase F, etc.) using the wild-type proteins you have obtained and then run a gel or mass spec to determine the M.W. after deglycosylation. Alternatively, you may just leave the glycans on if the mass from glycans are just several thousand daltons (how large is your protein?). Many crystal structures of secreted proteins are solved with the glycans on. They seem to help protein 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 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 patterns are much simpler. Other likely> candidates include > regular bakers' yeast and Hansenula polymorpha. I would> try both the native > and the mutant proteins in those hosts.> > There are tricks one can play with > Pichia to try and make it express the> deglycosylated protein but it almost > sounds like you've hit on something> very essential since you're going from > good expression down to no> expression.> > Lastly, you could try insect cells > :)> > Artem> > >> > 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> > nucleic acid sequence appears to be correct, > i.e. we have not introduced> > any unintentional frame shifts, stop codons, > or anything like that. Is> > this a common phenomenon? Are there any tricks > to get the Pichia to do> > its thing? Any chance that alternative > substitutions will work when Ala> > does not? Or are we better off (a) trying > to deglycosylate> > enzymatically, or (b) trying a different expression host? > All opinions> > and anecdotes welcome.> >> > Thanks!> > 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)> >> > _ Connect and share in new ways with Windows Live. http://www.windowslive.com/share.html?ocid=TXT_TAGHM_Wave2_sharelife_012008
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
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 could try a mammalian expression system as an alternative. I have been using Invitrogen's Flp-In system to generate stable cell lines in HEK293 Flp-In cells and has worked very well in my hands (I'm not affiliated with Invitrogen). Hope this helps. Vangelis On Mar 4, 2008, at 22:25, Radisky, Evette S., Ph.D. wrote: 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 nucleic acid sequence appears to be correct, i.e. we have not introduced any unintentional frame shifts, stop codons, or anything like that. Is this a common phenomenon? Are there any tricks to get the Pichia to do its thing? Any chance that alternative substitutions will work when Ala does not? Or are we better off (a) trying to deglycosylate enzymatically, or (b) trying a different expression host? All opinions and anecdotes welcome. Thanks! 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)
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
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 are enormous and heterogenous. That means that when you deglycosylate, you end up with a protein sample that has glycans of varying length which certainly does not help in crystallization. There are some commercial pichia strains out there with a modified glycosylation machinery. But I would certainly commend Artem's suggestion to switch to baculo. Cheers, Rob Meijers Synchrotron Soleil "Radisky, Evette S., Ph.D." <[EMAIL PROTECTED]> wrote: Removal of glycosylation sites in Picha expression construct 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 nucleic acid sequence appears to be correct, i.e. we have not introduced any unintentional frame shifts, stop codons, or anything like that. Is this a common phenomenon? Are there any tricks to get the Pichia to do its thing? Any chance that alternative substitutions will work when Ala does not? Or are we better off (a) trying to deglycosylate enzymatically, or (b) trying a different expression host? All opinions and anecdotes welcome. Thanks! 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) - Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now.
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
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 enzymatic treatment with either Endo H or PNGaseF (although you may need to denature the protein to get complete removal with the latter enzyme) I'm curious about your choice of mutations though. Although I know it's standard to perform Alanine substitution, switching from a known surface Asn to an Ala seems pretty drastic to me. If I perform a "google" survey of the literature it seems mutating the Asn to an Asp is an alternative (and better ?) choice, I also have seen publications where mutation of the Ser/Thr residue of the N-glycosylation recognition site to an Alanine appears to work. I also agree with others on the CCP4 bb of trying the mutations individually. 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 Radisky, Evette S., Ph.D. wrote: 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 nucleic acid sequence appears to be correct, i.e. we have not introduced any unintentional frame shifts, stop codons, or anything like that. Is this a common phenomenon? Are there any tricks to get the Pichia to do its thing? Any chance that alternative substitutions will work when Ala does not? Or are we better off (a) trying to deglycosylate enzymatically, or (b) trying a different expression host? All opinions and anecdotes welcome. Thanks! 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)
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
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 in Pichia). The protein has been previously produced and crystallized by another lab; in that case the double Ala mutant was expressed in CHO cells. It has been shown that glycosylation is not required for function. There are several reports of related (but natively nonglycosylated) proteins being produced in Pichia. We decided to try Pichia because we were already using it for another project, and we decided to introduce the Ala mutations because that mutant protein had apparently been crystallographically well behaved previously. Thanks again, 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)
Re: [ccp4bb] 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 Im 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
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
I don't know much about Pichia expression system. You can consult glycobiologists if you are 100% sure that no mistake was made during the experiments. Our lab has been using Drosophila S2 insect cell expression system. With this system, you can maintain cell culture using roller bottles or flasks (so better than the baculovirus expression system). The N-linked glycan is about 1000 dalton per site which can be removed easily by enzymes. The yield is typically above several mg per liter for even large proteins (>80 kDa). Holly > Date: Wed, 5 Mar 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 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 in Pichia). The protein has been previously > produced and crystallized by another lab; in that case the double Ala > mutant was expressed in CHO cells. It has been shown that glycosylation > is not required for function. There are several reports of related (but > natively nonglycosylated) proteins being produced in Pichia. We decided > to try Pichia because we were already using it for another project, and > we decided to introduce the Ala mutations because that mutant protein > had apparently been crystallographically well behaved previously. > > Thanks again, > 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) _ Helping your favorite cause is as easy as instant messaging. You IM, we give. http://im.live.com/Messenger/IM/Home/?source=text_hotmail_join
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
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
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
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 P: 512-471-4778, F: 512-232-3420 [EMAIL PROTECTED]
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
If you have poor conservation in this region and the region itself is a loop, I would perhaps attempt to replace the entire loop with another one - from a homologue that does not have the glycosylation site(s). Not that this is guarranteed to work, but it might. Artem -Original Message- 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. 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 in Pichia). The protein has been previously produced and crystallized by another lab; in that case the double Ala mutant was expressed in CHO cells. It has been shown that glycosylation is not required for function. There are several reports of related (but natively nonglycosylated) proteins being produced in Pichia. We decided to try Pichia because we were already using it for another project, and we decided to introduce the Ala mutations because that mutant protein had apparently been crystallographically well behaved previously. Thanks again, 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)
