Methods for producing substantially homogeneous hybrid or complex n-glycans in methylotrophic yeasts

[0092]Reagents used in Example 2 included antibiotics, such as Blasticidin S (Fluka), Zeocin (Invitrogen), Nourseothricin (Werner BioAgents), Geneticin G418 (Invitrogen), and Hygromycin B (Calbiochem); Bacto Agar (Difco), Bacto peptone (Difco), Bacto yeast extract (Difco), Biotin (Sigma), BMGY (see REAGENT SETUP), BMMY (see REAGENT SETUP), Citric acid (Calbiochem), Deionized water (dd-water), DTT (Sigma), Glucose monohydrate (Merck), Glycerol (Biosolve), HEPES (Sigma), Methanol (Biosolve), NaCl (Merck); restriction enzymes, such as AvrII (New England Biolabs), BsiWI (New England Biolabs), BstBI (New England Biolabs), PmeI (New England Biolabs), SapI (New England Biolabs), Sorbitol (Sigma), YNB (yeast nitrogen base) without amino acids (Difco) and YPD media and plates (see REAGENT SETUP).

TABLE 2AntibioticsAntibioticFinal concentration (μg / ml)Blasticidin500Zeocin100Nourseothricin100G418350Hygromycin B150

[0093]Equipment

[0094]Equipments used in Example 2 included 24-well culture plates (

EXAMPLE 3

Results

[0244]The workflow presented in FIGS. 1a and 1d allows engineering of the N-glycosylation pathway of any wild type P. pastoris strain. The construction of a strain that modifies its glycoproteins with Gal2GlcNAc2Man3GlcNAc2 N-glycans requires the consecutive integration of five GlycoSwitch vectors into the Pichia genome. Each of these plasmids contains a different dominant antibiotic resistance marker for selection. As a consequence, it is critical that the starting strain is still sensitive to all five antibiotics: blasticidin, zeocin, hygromycin, geneticin and nourseothricin. Some other combinations of engineering enzyme—selection marker are available (see Table 4), but not all. One can start from the GS115 wild type strain because its histidine auxotrophy provides an additional selection maker that allows selection for integration of a pPIC9-derived vector that drives the production of a protein of interest.

[0245]This Example describes results of three mouse protein