Key words: monoclonal antibody/N-glycosylation/transgenic plants
Transgenic plants are an important system for the expression of therapeutic recombinant proteins (Moffat, 1995). Hiatt et al. (1989) demonstrated that the coexpression in tobacco of light and heavy immunoglobulin chains results in the production of functional antibodies. Since then, a number of groups have expressed antibodies in plants, either to modify plant performance or to exploit plants as bioreactors for large-scale production of full-length IgG (During et al., 1990; Ma et al., 1994) and secretory antibodies (Ma et al., 1995).
It has been shown that some of the properties of immunoglobulins depend on their glycosylation. In general, there is one conserved N-glycosylation site per heavy chain of IgG in the CH2 domain constitutive of the Fc region (Rademacher et al., 1986). Glycosylation of the Fab region has also been described in about 30% of serum antibodies (Rademacher et al., 1986). The N-glycosylation of the Fc contribute to the structural stability of the immunoglobulin. The two constitutive oligosaccharides of an IgG molecule stabilize the Fc by filling the interstitial region between the two CH2 domains (Parekh et al., 1985) and exert a subtle influence on protein tertiary and quaternary structures that is essential for activity. Aglycosylated IgG usually shows higher sensitivity to proteases and a loss of binding capacity to monocyte Fc receptors (for recent reviews, see Jefferis and Lund, 1997; Wright and Morrison, 1997). As a consequence, the N-glycosylation of recombinant antibodies is one of the key steps for the production of fully functional immunoglobulins by an expression system.
The N-glycosylation in higher organisms is conserved but differs slightly in detail. The processing of the N-linked glycans occurs along the secretory pathway as the glycoprotein moves from the endoplasmic reticulum through the Golgi apparatus to its final destination. Glycosidases and glycosyltransferases located in the Golgi apparatus successively modify the oligosaccharide precursor to high-mannose-type N-glycans and then into complex-type N-glycans. The complex-type N-glycans arise from the transfer in the Golgi apparatus of monosaccharide residues onto the core Man3GlcNAc2 under the action of several glycosyltransferases. Since some of these modifications are specific for the expression system, the structure of mature complex-type N-glycans associated with plant, insect, yeast, or mammalian glycoproteins will differ. Thus, no heterologous system will be able to reproduce mammalian glycans exactly. Plants, insects, and yeast do not introduce sialic acid on their glycoproteins and synthesize N-glycans having carbohydrate motifs that are not found in mammals. For instance, in plants, complex-type N-glycans are characterized by the presence of [beta](1,2)-xylose residue linked to the [beta]-mannose and/or an [alpha](1,3)-fucose residue, instead of an [alpha](1,6)-fucose residue, linked to the proximal glucosamine (for a recent review, see Lerouge et al., 1998). Since plants are gaining acceptance for the expression of recombinant therapeutic proteins, it is important to examine in detail to what extent glycans of mammalian glycoproteins produced in transgenic plants differ from original ones.
The monoclonal antibody (MAb) Guy's 13 is a mouse IgG1 class antibody, which recognizes a cell-surface protein of Streptococcus mutans, the bacteria which is the principal cause of dental caries in humans (Smith and Lehner, 1989). This MAb contains two potential N-glycosylation sites on its constitutive heavy chain. In addition to the highly conserved glycosylation site in the Fc region, MAb Guy's 13 has a second Asn-74-Ser-Ser N-glycosylation consensus sequence located in the Fab part of this IgG1 molecule. A full-length MAb Guy's 13 was expressed in tobacco (Ma et al., 1994). This plant MAb or plantibody was found to be functional in terms of antigen recognition and binding. In this article, we report a detailed structural comparison of the N-glycans of the MAb Guy's 13 expressed in mouse and in tobacco plants using immunochemical and physicochemical approaches. N-Glycosylation analysis of the mouse Guy's 13 antibody
The MAb Guy's 13 has two potential N-glycosylation sites located on the heavy chain: the conserved site in the Fc fragment and an additional site on Asn-74 located on the Fab fragment. The structure of the N-linked glycans and their distribution on the two potential N-glycosylation sites were first investigated by affinodetection on blots using Ricinus communis agglutinin (RCA). This lectin specifically binds to N-acetyllactosamine sequence usually found in mammalian complex-type N-glycans. Heavy and light chains constitutive of the mouse IgG and the Fab fragment were separated by SDS-PAGE in reducing conditions, and they were affinodetected on blots (Figure
Figure 1. SDS-PAGE and Western blot analysis of the mouse MAb Guy's 13 and Fab fragment. The purified mouse MAb Guy's 13 (a) and Fab fragment (b) were analyzed by SDS-PAGE in reducing conditions and detected by silver staining in the gel (lanes 1) or by affinodetection with RCA of the corresponding blots (lanes 2). [gamma], Guy's 13 heavy chain; [kappa], Guy's 13 light chain.
The monosaccharide analysis of the mouse MAb was performed in two steps. The neutral and amino sugars were released by hydrolysis with 2 M TFA. The identification and quantification of monosaccharides were done by comparison to calibrated standards as previously reported (Hardy, 1989). Fuc, Gal, GlcNAc, and Man were detected in the molar ratio 0.65:1.5:3.8:3.0. Neuraminic acid released in mild acidic conditions (0.2 M TFA) was estimated to represent about 10% of the sugar content (Anumula and Taylor, 1991). The structures of N-linked glycans attached to the mouse MAb were determined by chromatographic analysis and nuclear magnetic resonance (NMR). The oligosaccharides were released from the MAb with PNGase F and analyzed by HPAE-PAD chromatography as reported in Rohrer et al. (1995). The mouse MAb N-linked glycans were found to be separated into neutral and sialylated oligosaccharides in a 9:1 ratio, confirming that sialylated N-linked glycans represent about 10% of the total oligosaccharides(Figure
Figure 2. Chromatography of oligosaccharides released from the mouse MAb Guy's 13. (A) HPAEC-PAD on CarboPac PA1 column of the mouse MAb Guy's 13 N-glycans. (B) HPLC profile on a C18 reverse-phase column of PA-oligosaccharides isolated from the mouse MAb Guy's 13. N-linked glycans released from the mouse MAb with PNGase F were reductively aminated with 2-aminopyridine and separated by reverse-phase HPLC as described in Materials and methods. Peaks a to d were identified as the pyridylamino derivatives of oligosaccharides A to D, respectively,presented in Table I.
Table I. N-Glycosylation analysis of the plantibody Guy's 13
The plantibody Guy's 13 was purified from mature transgenic tobacco plants by several affinity chromatography steps. A preliminary analysis of the N-linked glycans attached to the plantibody Guy's 13 was obtained by affino- and immunodetection on blots using glycan-specific probes (Figure
Figure 3. SDS-PAGE and Western blot analysis of the plantibody Guy's 13. The affinity-purified plantibody Guy's 13 was analyzed by SDS-PAGE in reducing conditions and protein silver staining in the gel (lane 1), or on the corresponding blots by affinodetection with concanavalin A (lane 2) or by immunodetection with antibodies specific for [alpha](1, 3)-fucose (anti-fucose antibodies, lane 3) and for [beta](1, 2)-xylose (anti-xylose antibodies, lane 4) epitopes of plant N-linked glycans.
Figure 4. Immunodetection of complex-type plant N-glycans on Fab and Fc fragments. (A) Immunoblot of plantibody Guy's 13 and papain digested fragments under nonreducing conditions. Three panels are shown, in which detection was with (a) anti-heavy chain, (b) anti-light chain, and (c) anti-xylose antibodies. Lanes 1, undigested plantibody Guy's 13 (4 µg); lanes 2, papain digested plantibody Guy's 13 (4 µg); lane 3, undigested mouse MAb Guy's 13 (4 µg); and lane 4, papain digested mouse MAb Guy's 13 (4 µg). (B) ELISA analysis of the plantibody Guy's 13 and of the corresponding Fab fragment bound to streptococcal antigen. Plantibody Guy's 13 and Fab fragment were incubated in streptococcal antigen coated plates and detected after binding with either anti-heavy chain (anti-Fc fragment) (1:16 dilution), anti-light chain (1:128 dilution) or anti-xylose (1:16 dilution) antibodies. Columns 1, undigested plantibody Guy's 13; columns 2, papain digested plantibody Guy's 13; columns 3, papain digested plantibody purified by incubation with protein G Sepharose beads; columns 4, TBS instead of plantibody or plantibody fragments.
The distribution of plant N-glycans on the two potential N-glycosylation sites of the plantibody Guy's 13 was investigated by Western-blot and ELISA analysis (Figure
Figure 5. HPAE-PAD chromatography of high-mannose type N-glycans isolated from the plantibody Guy's 13. High-mannose-type N-glycans were isolated by affinity chromatography on a concanavalin A-Sepharose column and then analyzed by HPAEC-PAD. The different oligosaccharides were identified by comparing their retention times to standard oligosaccharides isolated from mammalian and plant sources. The structures of Man-5 to Man-8 oligosaccharides are presented in Table II.
Figure 6. HPAE chromatography of pyridylamino derivatives of complex-type N-glycans isolated from the plantibody Guy's 13. N-linked glycans that were not retained on immobilized concanavalin A, were coupled with aminopyridine and then analyzed by HPAEC combined to a fluorescence detection. (A) HPAEC profile of PA-oligosaccharides obtained by derivation of N-linked glycans isolated from the plantibody Guy's 13. (B) HPAEC profile of the PA-oligosaccharide obtained after digestion with Jack bean [beta]-N-acetylglucosaminidase. Peaks a, b, and c were identified as the pyridylamino derivatives of oligosaccharides A, B, and C, respectively, presented in Table II.
After hydrolysis with 2 M TFA of 50 µg of the plantibody, Fuc, Xyl, GlcNAc, and Man were detected by HPAEC-PAD in the molar ratio 0.5:0.5:3.0:5.0. The glycans N-linked to the heavy chain were released from the whole plant recombinant immunoglobulin by sequential digestion with pepsin and PNGase A as previously reported (Tomiya et al., 1987; Rayon et al., 1996). PNGase A is a plant peptide N-glycosidase which is able to release all N-linked glycans including oligosaccharides having a fucose residue [alpha]-linked to the O-3 of the proximal glucosamine. The N-glycans were then purified by elution through a AG50 X2 and a C18 columns, desalted on a Bio Gel P4 column and analyzed by HPAE-PAD chromato-graphy (not shown). The HPAEC-PAD profile has shown a mixture of N-linked glycans which molar ratio were quantified by integrating the amperometric signals detected by HPAEC-PAD. To fully identify the structure of the different oligosaccharides, the mixture of N-glycans released from the plantibody Guy's 13 was fractionated by affinity chromatography on a concanavalin A-Sepharose 4B column in order to separate high-mannose-type N-glycans retained by affinity, from unretained complex-type N-glycans. The fraction containing the high-mannose-type N-glycans was analyzed by HPAE-PAD chromatography (Figure
Figure 7. Electrospray-mass spectrometry of the PA-derivatives of the xylose- and fucose-containing N-glycans isolated from the plantibody Guy's 13. Ions a, b, and c correspond to the (M+Na)+ ions of the pyridylamino derivatives of plant N-glycans A, B, and C presented in Table II. b* and c* are the double charge (M+2Na)2+ ions of oligosaccharides B and C.
Table II.
Several mammalian proteins have been successfully produced in plants and some of them are indistinguishable from those produced in mammalian cells as far as amino acid sequence, conformation and eventually biological activity are concerned (Owen and Pen, 1996). However, most clinically important mammalian proteins are glycosylated and their glycosylation affects their physiochemical properties, including resistance to protease attack and solubility. Glycans N-linked to therapeutic glycoproteins are also responsible for some of their biological functions such as antigenicity, immunogenicity, or plasma clearance rate. In a field where the utilization of plants as expression systems for the production of therapeutic recombinant proteins starts to be important, it is urgent to explore the capacity of plant cells to produce and glycosylate mammalian glycoproteins. Our detailed analysis of the N-glycosylation pattern of the MAb Guy's 13, counted with previous results on the biological activity of this MAb expressed in transgenic tobacco (Ma et al., 1994), constitutes the first complete comparative study of mammalian glycoprotein produced in a transgenic plant system.
Four different oligosaccharide structures were found N-linked to the mouse MAb Guy's 13 and were identified as complex-type N-glycans containing [alpha](1,6)-fucose at their innermost N-acetyl-glucosamine residue and galactose and sialic acid to different extents. These results are consistent with previous structural studies of mouse IgG N-glycans (Rademacher et al., 1986; Mizuochi et al., 1987; Rothman et al., 1989; Rohrer et al., 1995). The N-glycosylation analysis of the plantibody Guy's 13 was carried out on material purified from mature tobacco plants by affinity chromatography on both protein G and immobilized anti-mouse IgG1 antibodies. The diversity of oligosaccharide structures on the plantibody Guy's 13 is far higher than on murine MAb. Indeed, we have identified eight different oligosaccharides on the plantibody molecule representing an array of structurally related oligosaccharides from high-mannose-type N-glycans (40%) to modified glycans (60%). In conclusion, the heterogeneity of the carbohydrate moiety in the antibody Guy's 13 and consequently the number of Guy's 13 glycoforms is higher in transgenic tobacco than in mouse. The plant N-linked glycan structures A, B, and C containing [beta](1,2)-xylose and the [alpha](1,3)-fucose residues linked to the core Man3GlcNAc2 (Table II) were previously found in many plant glycoproteins. Furthermore, the complex-type N-glycans B and C, the major oligosaccharides N-linked to the plantibody Guy's 13, are oligosaccharide structures usually found on plant extracellular glycoproteins. These oligosaccharides are known to be rapidly trimmed by the action of exoglycosidases when the glycoprotein is stored in plant vacuole giving the truncated Man3XylFucGlcNAc2 glycan A (Lerouge et al., 1998). The presence of such oligosaccharides with terminal N-acetylglucosamine residues is indicative of the secretion of most of the plantibody Guy's 13 expressed in transgenic tobacco, which confirms a previous study showing that plantibodies are secreted by plant cells and accumulates in the tobacco apoplasm (Ma and Hein, 1995). However, it cannot be excluded that the large structural heterogeneity of the glycans found on the plantibody could be the result of the storage of part of this recombinant IgG in other compartments of the plant secretory pathway. Indeed, storage of the plantibody in the endoplasmic reticulum or in the vacuole will result in N-glycans having unprocessed or truncated structures, respectively (Lerouge et al., 1998).
The MAb Guy's 13 contains two potential N-glycosylation sites on the heavy chain located on the Fc and the Fab fragments. Both sites of the mouse MAb Guy's 13 were found to be N-glycosylated by complex-type N-glycans. By ELISA and Western blot analysis of the full length or the papain digested plantibody, it was demonstrated that plant complex N-glycans are also localized on both the Fc and the Fab fragments of the plant IgG1. However, the precise distribution of plant N-linked glycans on both sites has not been fully determined, due to the limitations related to the small quantities of purified Fab and Fc fragments isolated from the plantibody. However, our results clearly illustrate that both the highly conserved N-glycosylation site in the CH2 domain of the heavy chain of murine IgG and the additional site (Asn-74) found on the Fab region of the Guy's 13 are occupied in both the murine and in the plant expression system.
In humans, the mouse MAb Guy's 13 when applied directly to the teeth can prevent colonization of oral cavity with Streptococcus mutans and reduce the risk of dental caries (Ma et al., 1990). The production of functional MAb in plants, for a low cost, should provide the large amount of immunoglobulin required for applications in the area of topical immunotherapy. Plant specific glycosylation is sufficient to produce soluble and biologically active IgG. However, the oligosaccharide side chains of immunoglobulins have many other functions than 'spacers" between the two CH2 domains. The IgG N-glycans are necessary for effector functions of the antibodies such as recognition by receptors that mediate their survival in the circulation, binding to macrophage Fc receptors, complement fixation or elimination of antigen-antibody complexes (Wright and Morrison, 1997). Further studies will help to answer whether or not plantibody N-glycans will also assume some of these functions in vivo.
Differences that exist in the glycosylation patterns in plants and mammals could represent an important limitation to the use of recombinant mammalian glycoproteins produced in transgenic plants for in vivo therapy. In this study, the comparison of murine and plantibody Guy's 13 clearly illustrates these differences in the glycosylation of the same protein backbone. Some similarities exist between complex-type N-glycans from both expression systems such as the presence of a common Man3GlcNAc2 core substituted by terminal GlcNAc residues. However, additional motifs on this core, i.e., the [alpha](1,3)-fucose and the [beta](1,2)-xylose residues, can be highly immunogenic in mammals. In the context of large quantities of antibodies that may be introduced into the human organism for some therapies, a sensitization to the plant complex-type N-glycans identified on the plantibody Guy's 13, might occur. As a consequence, for some in vivo applications of recombinant antibodies produced in transgenic plants, strategies have to be developed to obtain plantibodies with carbohydrate profiles structurally more consistent with ones obtained from mammalian cells. Materials
Ricinus communis agglutinin (RCA) coupled with horseradish peroxidase, pepsin and [beta]-N-acetylglucosaminidase from Jack bean were from Sigma. PNGase A and F were purchased from Boehringer Mannheim. Man-5 (RNase B), Man-6 (RNase B), Man-7(II) (bean phaseolin), Man-8 (bean phytohemagglutinin), and Man-9 (soybean agglutinin) were isolated from mammalian and plant glycoproteins and identified by proton NMR. The pyridylamino derivative standards of plant N-glycans were prepared by labeling with 2-aminopyridine (Hase et al., 1984) complex oligosaccharides isolated from bean phytohemagglutinin (Rayon et al., 1996) and from Arabidopsis thaliana glycoproteins. Purification of the plantibody Guy's 13
Mature transgenic plants expressing Guy's 13 MAb were homogenized in 1.5 volume of Tris-buffered saline (TBS, 75 mM NaCl and 10 mM Tris-HCl, pH 8). After centrifugation of the homogenate at 12,000 × g for 30 min at 4°C, proteins were precipitated from the supernatant in the presence of ammonium sulfate (50% saturation). After a second centrifugation in the same conditions as above, the protein pellet was solubilized in TBS. The proteins were then submitted to a second precipitation with ammonium sulfate (50% saturation). The second protein precipitate was resuspended in TBS and the solution was passed through a 0.45 µm filter. IgG was purified from this protein extract by affinity chromatography on a protein G-agarose column (MAbtrap GII, Pharmacia Biotech) followed by a further affinity purification step using Sepharose 4B beads coupled to goat anti-mouse IgG1 antibodies (Sigma UK). SDS-PAGE and immunoblotting experiments
The antibody Guy's 13 was separated in a 15% SDS-PAGE in both reducing and nonreducing conditions and was then transferred onto a nitrocellulose membrane. Immunodetections with anti-xylose or anti-fucose antibodies were performed according to Faye et al. (1993). Affinodetection of high-mannose-type N-glycans was carried out using the concanavalin A/peroxidase method (Faye and Chrispeels, 1985). Affinodetection by RCA was carried out as reported in Fitchette-Lainé et al. (1998). Papain digestion of plantibody Guy's 13
Purified plantibody Guy's 13 was incubated with papain (1:100) (Sigma, UK) in 0.1 M Tris-HCl pH 7 containing 2 M EDTA and 20 mM cysteine, for 16 h at 37°C. The reaction was stopped by incubation with 0.1 M iodoacetamide for 1 h at 37°C. Con-taminating Fc was removed by incubation with protein G Sepharose beads (Pharmacia Biotech, UK) for 5 h at 37°C, and the supernatant was recovered by centrifugation. ELISA analysis of plantibody and Fab fragment bound to streptococcal antigen
ELISA plates (Dynatech, Immulon) were coated with streptococcal antigen at a predetermined optimal concentration of 2 µg/ml. After blocking with 5% nonfat dry milk in TBS containing 0.01% Tween 20, plantibody solutions were applied for 2 h at 37°C. The plates were washed with TBS containing 0.05% Tween 20, and either anti-murine kappa (Bradsure Biologicals, UK), anti-murine gamma (anti-Fc fragment) (Sigma, UK) or anti-xylose antibodies were applied for 2 h at 37°C. After further washings, the next incubation was done with an appropriate alkaline phosphatase-conjugated antiserum for 2 h at 37°C. Finally, detection was performed using disodium p-nitrophenylphosphate (Sigma, UK). Absorbance was read at 405 nm. Monosaccharide analysis
Ten micrograms of mouse or plantibody Guy's 13 were hydrolyzed with 2 M TFA at 105°C for 2 h. The samples were dried out, and the analysis of monosaccharides was carried out by HPAEC-PAD as previously reported (Hardy, 1989). Sialic acids were released from the mouse MAb with 0.2 M TFA at 80°C for 1 h. The analysis and quantification of the sialic acids was done by HPAEC-PAD according to Anumula and Taylor (1991). Isolation of N-linked glycans from the mouse MAb
The purified mouse Guy's 13 (3 mg) was digested with trypsin (1% w/w) in a 50 mM ammonium carbonate buffer, pH 8, for 18 h at 37°C. The solution was then heated at 100°C for 5 min and treated with PNGase F (10 mU) for 24 h at 37°C. The oligosaccharides were purified by successively passing through a 2 ml AG50 X2 column and a C18 Sep-Pak cartridge. The oligosaccharides were finally desalted on a 40 × 1 cm Bio Gel P4 column. Isolation of N-linked glycans from the plantibody
N-linked glycans were released from 2 mg of purified plantibody Guy's 13 by sequential digestions with pepsin and PNGase A (1 mU) as previously reported (Tomiya et al., 1987; Rayon et al., 1996). The resulting free glycans were desalted on a Bio Gel P4 column. Complex-type N-glycans were then separated from the high-mannose-type N-glycans by affinity chromatography on a concanavalin A-Sepharose 4B column as reported previously (Montreuil et al., 1986). Analysis of N-linked glycans
HPAE-PAD chromatographies of oligosaccharides released from the mouse MAb and high-mannose-type N-glycans isolated from the plantibody were achieved on a Dionex DX 500 system equipped with a GP 50 gradient pump and a CarboPac PA1 column. Oligosaccharides were separated using a linear gradient from 0 to 100 mM NaOAc in 100 mM NaOH at 1 ml/min over 30 min. High-mannose-type N-glycans from the plant MAb were identified by comparing their retention times to standard oligosaccharides isolated from RNase B (Man-5 and Man-6), bean phaseolin (Man-7(II)), bean phytohemagglutinin (Man-8), and soybean agglutinin (Man-9) as previously reported (Rayon et al., 1996). Complex-type N-glycans were coupled with aminopyridine as described by Hase et al. (1984). Pyridylamino derivatives (PA) of the N-linked glycans isolated from the mouse MAb were separated by reverse-phase HPLC on a Spherisorb ODS 2 C18 column (250 × 4.6 mm). Elution and identification of PA-oligosaccharides were performed as described in Tomiya et al. (1988). The quantification of the molar ratio of the N-linked glycans isolated from the mouse MAb was deduced by integrating the fluorescence signals of PA-derivatives separated by reverse-phase HPLC. HPAE chromatography of the PA-derivatives of complex-type N-glycans isolated from the plantibody was achieved as described above for the separation of reducing oligosaccharides. However, PA-oligosaccharides were detected using a Spectra System FL 2000 (Spectra-Physics) fluorescence detector with excitation and emission wavelength at 320 and 400 nm, respectively. The sample was dissolved in 0.1 M NaOH before injection. Exoglycosidase treatment of the PA-oligosaccharides was carried out with 1 U of [beta]-N-acetylglucosaminidase in a 10 mM NaOAc buffer, pH 5, at 25°C for 18 h. The quantification of the molar ratio of the N-linked glycans isolated from the plantibody was assessed by integrating the amperometric signals detected by HPAEC-PAD of the mixture of complex and high-mannose-type N-glycans. Electrospray mass ionization
Electrospray mass ionization was performed on a quadrupolar mass spectrometer NERMAG R 1010U equipped with an Analytica of Branford atmospheric pressure electrospray source (Quad Servive, Poissy, France). The mass on charge (m/z) range was of 2000. The sample was dissolved in a 1:1 H2O:CH3OH solution containing 0.05% CH3COOH and infused into the electrospray ion source at a flow-rate of 1.5 µl/min by a Harvard syringe pump (Harvard Apparatus). The ion source temperature was 80°C (drying gas). Full scan spectra were acquired in the range of 500-1950 at a scan speed of 9s.
This work has been achieved in the frame of the French network 'GT-rec" supported by MENRT (ACC SV 14, no. 9514111) and CNRS (Program PCV). This work was also supported by the University of Rouen, the Region Haute-Normandie, the Wellcome Trust Grant 038403, and the European Community (BMH4-CT-97-2345). Marion Cabanes is recipient of a Biopole fellowship.
HPAEC-PAD, high pH anion exchange chromatography coupled to pulsed amperometric detection; MAb, monoclonal antibody; 1H NMR, proton nuclear magnetic resonance; PA, pyridylamino derivative; plantibody, recombinant antibody produced in transgenic plants; PNGase A, peptide N-glycosidase A; PNGase F, peptide N-glycosidase F; RCA, Ricinus communis agglutinin.
3To whom correspondence should be addressed
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
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