José A. Cremata, Raquel Montesino,. Omar Quintero, and Rossana GarcÃa. 1. Introduction. The methylotrophic yeast Pichia pastoris has been widely exploited ...
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8 Glycosylation Profiling of Heterologous Proteins José A. Cremata, Raquel Montesino, Omar Quintero, and Rossana García 1. Introduction The methylotrophic yeast Pichia pastoris has been widely exploited for its high-level expression of heterologous proteins by recombination of gene sequences of interest with the methanol-inducible alcohol oxidase gene (AOX1) promoter (1–3). Secreted and cytoplasmic expression of heterologous proteins at levels equivalent to Escherichia coli and significantly higher than in Saccharomyces cerevisiae has been achieved (4). In addition, P. pastoris cultures can be easily scaled up to high cell densities, and as a result, yields are also high on a volumetric basis (e.g., 12 g/L, for tetanus toxin fragment C [5], 2.5 g/L for invertase [6], 2.5 g/L for _-amylase [7]). Secreted products can comprise more than 80% of the protein in the medium (6). However, secretion is a complex process that is not only dependent on gene dosage, but also on other factors, such as signal sequence recognition and processing, proteolysis, and glycosylation. With regard to glycosylation, heterologous proteins secreted from S. cerevisiae are often hyperglycosylated with outer chains of mannose units of up to 50–150 residues added at N-asparagine-linked sites, which makes such glycoproteins highly antigenic and, therefore, unsuitable for use as human therapeutic drugs (8). To investigate glycosylation in P. pastoris, Tschopp et al. expressed the S. cerevisiae SUC2 gene in P. pastoris and examined in detail the structure of N-linked oligosaccharides added to its product, invertase (6). They observed that S. cerevisiae invertase secreted from P. pastoris is not hyperglycosylated, but contains outer chains of only 8–14 mannose residues (Man8–14), compared to an average length of >50 when the same enzyme is secreted from S. cerevisiae (9). The P. pastoris-secreted invertase resembles in size the endoplasmic reticulum core-glycosylated form of invertase seen in S. From: Methods in Molecular Biology, Vol. 103: Pichia Protocols Edited by: D. R. Higgins and J. M. Cregg © Humana Press Inc., Totowa, NJ
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cerevisiae sec18 mutants at nonpermissive temperature (9). The core-like structure of the oligosaccharides was confirmed by Trimble et al. using NMR techniques (10,11). They found that 75% of the oligosaccharides on P. pastoris secreted invertase are Man8–9, and most of the remaining oligosaccharides are Man10–11. The structures are all typical of core glycosylation structures on fungal proteins (11). Of potential importance to the use of P. pastoris glycoproteins as human pharmaceuticals, the oligosaccharides in P. pastoris-secreted invertase lack terminal _1,3-mannose residues that are commonly found on proteins secreted from S. cerevisiae and are known to be highly antigenic (10,11). However, the suitability of foreign glycoproteins expressed in P. pastoris for pharmaceutical use remains problematic, since the lower eukaryotic structure of P. pastoris oligosaccharides is significantly different than that of mammalian cells. Unfortunately, little information exists on oligosaccharide structures on other glycoproteins secreted from P. pastoris. Although some proteins appear to have only short oligosaccharide structures like invertase, others appear to be hyperglycosylated (4,12). The variability of carbohydrate structures on glycoproteins secreted from P. pastoris makes their analysis an important aspect of protein characterization in this yeast. As a step in this analysis, oligosaccharide “profiling” methods have been developed in recent years. One of them, called FACE for fluorophore-assisted carbohydrate electrophoresis, is based on the reductive amination of glycans by 8-amino-1,3,6-naphthalene trisulfonic acid (ANTS) and their separation by polyacrylamide gel electrophoresis (13). More recently, a second method based on the resolution of the same ANTS–oligosaccharide derivatives by HPLC analysis has been described (14). By a twodimensional analysis process involving HPLC retention times in one dimension and relative migration index (RMI) during electrophoresis in the other, oligomannoside structures can readily be determined. These profiling methods, which are described in this chapter, greatly simplify the work of characterizing carbohydrates present on natural and recombinant glycoproteins. 2. Materials 2.1. Chemicals and Enzymes All reagents should be of analytical grade. Recommended sources of specialty chemicals and enzymes include: ANTS from Molecular Probes (Eugene, OR); N-glycosidase F (PNGase F) from either Boehringer Mannheim (Indianapolis, IN) or New England BioLabs (Beverly, MA); and _-mannosidase from Aspergillus saitoi (an exoglycosidase) from Oxford GlycoSystems (Abingdon, UK).
2.2. Buffers, Stock Solutions, and Equipment 1. Enzyme incubation buffer: 0.2 mM sodium phosphate buffer, pH 8.6. 2. Denaturing solution: 2.5% SDS, 1.5 M `-mercaptoethanol.
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3. ANTS solution: 0.15 M ANTS in an acetic acid/water solution (3:17 v/v). Gentle warming in a 60°C bath is required to dissolve the ANTS completely. This solution may be stored at –70°C. 4. Sodium cyanoborohydride solution: 1.0 M in dimethyl sulfoxide (DMSO). This solution must be made fresh daily. 5. Gel electrophoresis apparatus: Mighty Small SE250, Hoeffer Scientific Instruments (San Francisco, CA). 6. Acrylamide stock solution: an aqueous solution of 60% (w/v) acrylamide, 1.6% (w/v) N, N'-methylene-bis-acrylamide. 7. 4X stock of gel buffer: 1.5 M Tris-HCl, pH 8.5. 8. 10X stock of electrophoresis buffer: 1.92 M glycine, 0.25 M Tris base, pH 8.5.
3. Methods 3.1. Release of Oligomannosides from Proteins 1. Dialyze a 50–250-μg sample of glycoprotein against distilled water, and then dry the sample in a centrifugal vacuum evaporator (see Note 1). 2. Dissolve the dried glycoprotein sample in 30 μL of enzyme incubation buffer. 3. Add 2 μL of denaturing solution, and heat at 100°C for 5 min. 4. Cool the sample to room temperature and add 5 μL of a 7% (v/v) solution of Nonidet P-40. 5. Add 5 μL of PNGase F, mix well, and centrifuge briefly to bring the entire sample to the bottom of the tube. 6. Incubate the sample at 37°C for 16 h. 7. Centrifuge the sample at 10,000g for 5 min to clear the solution. Discard the pellet. 8. Add 130 μL of cold ethanol, and incubate for 1 h in an ice bath or for 20 min at –20°C. 9. Centrifuge at 10,000g for 3 min. 10. Carefully separate the supernatant, which contains the oligosaccharide pool, from the pellet. 11. Dry the protein pellet from step 9 in a centrifugal vacuum evaporator for 10 min. Resuspend it in 50 μL of water, and add 3 vol of cold ethanol. Incubate for at least 1 h in an ice bath or for 20 min at –20°C. 12. Centrifuge at 10,000g for 3 min, and add the supernatant from step 10 (see Note 2). 13. Dry the oligosaccharide pool in a centrifugal vacuum evaporator for at least 1 h with low heating. Do not exceed 45°C during evaporation. If the ANTS derivatization reaction is not to be performed immediately, store the pellet at –20°C.
3.2. ANTS Derivatization of Oligosaccharides 1. Add 5 μL of ANTS solution to the dry oligosaccharide pool sample described in Subheading 3.1., step 13. 2. Add 5 μL of the cyanoborohydride solution, and mix well. Be sure that all the oligosaccharides are in solution. 3. Centrifuge briefly to bring sample to the bottom of the tube, and incubate the sample for 16 h at 37°C or 2 h at 45°C (see Note 3).
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4. Dry the reaction product in a centrifugal vacuum evaporator for at least 1 h with low heat (
