Frequently Asked Questions: Improvements
Q: Does the secretion efficiency change in the engineered strains?
A: The effect of glycan engineering on secretion efficiency has to be analyzed on a case-by-case study, depending on the heterologous protein expressed. Moreover, this analysis can be difficult to do, especially when the wild-type strain adds hyperglycosyl structures to the heterologous protein, resulting in a smearing of the protein signal onto an acrylamide gel or immunoblot (e.g.: Is an increased signal in a GlycoSwitch strain the result of higher secretion efficiency, or merely due to reduced heterogeneity of the produced protein?). In the near future, some extra data will be added concerning the effect of glycan engineering on the secretion of a few “test” proteins.
Q: Why OCH1 gene knock out by knock in?
A: The knock-in procedure is very efficient (typically, the construct is targeted correctly to the OCH1 locus in 50% of the primary selected clones), in contrast to double homologous recombination-based OCH1 knock out, where cumbersome selection procedures and screening of a large number of clones is required. The high efficiency of our knock-in strategy allows the conversion of any existing expression strain into a GlycoSwitchM5 strain, thus saving the user much of the re-optimization of protein
expression.
Q: Are the systems functioning equally well under both the AOX1 and GAP promotor systems?
A: Generally, yes. But if your secreted glycoprotein is under control of the GAP promoter you can only use the GAP-controlled GlycoSwitch system. Additionally, we advise using the system under control of AOX1 promoter if your glycoprotein of interest is expressed from this promoter.
Q: What about the O-glycans?
A: To the best of our knowledge, the O-glycans are not affected by our GlycoSwitch strategy. This has, however, not been analyzed thoroughly.
Q: What are the other remaining peaks in the profiles?
A: The few percent of non-Man5GlcNAc2 glycans have not been identified (this is very difficult due to their low abundance) but they presumably are alpha-1,2-mannosidase digestion intermediates.
Q: Are the strains stable? Do the strains need further antibiotic selection?
A: All constructs for glycan engineering are stably integrated into the Pichia genome via a single homologous recombination event. Transformants are isolated on selective medium. To our knowledge however, no further selection is needed once the suitable glycosylation strains have been isolated.
Q: Do the strains need adapted growth media?
A: No, the strains can be grown in the usual media like YPD, BMGY, BMMY, etc. The genomic integration of the plasmids is sufficiently stable, so no antibiotics need to be added.
Q: Are the strains suited for expression of protein vaccines?
A: The strains can be used for such purposes. GlycoSwitch strains no longer perform hyperglycosylation on secreted proteins, hence increasing the homogeneity of the end product. The latter is of importance when wanting to use the recombinant protein for pharmaceutical approaches.
Q: Do the strains grow well?
A: Although disruption of OCH1 in S. cerevisiae results in a crippled strain, no such phenotype was observed when inactivating the OCH1 gene in Pichia pastoris via the described knock-in strategy. GlycoSwitch strains tend to grow perfectly well at 30oC.
Q: Are the strains leaking proteins?
A: In some cases we find traces of alpha-1,2-mannosidase in the medium. Whether this due to “real” leakage or is a result of cell lysis is unknown.
Q: What’s the best way to analyze the N-glycans of our secreted proteins?
A: We recommend using carbohydrate electrophoresis on a DNA-sequencer (Callewaert et al. Glycobiology 11 (2001), 275-281). This method has been used successfully to evaluate strain glycosylation profiles (Vervecken et al. Appl. Environ. Microbiol. 70 (2004): 2639-2646). Detailed protocols are available from the developers. This analysis is also offered as a contract service by the laboratories of the developers. Typically, 1-2 microgram of the purified protein is sufficient.
Q: Are the strains available to the general public?
A: The strains are available to only to licensees or academics by material transfer agreement.
Q: Are the new strains pathogenic?
A: No.
