Online Resources Online Resource 1 Schematic non

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Online Resources
Online Resource 1 Schematic non-proportional representation of the T-DNA region coding for binary vectors
patagHB-HC (upper) and patagHB-LC (lower). Pphas/ UTR arc: promoter of the β-phaseolin gene and 5´
untranslated region of the arc5-I gene; 2S2: sequence coding for the signal peptide 2S2 seed storage protein from
Arabidopsis thaliana; HC: Sequence coding for the heavy chain of anti-HBsAg monoclonal antibody; LC: Sequence
coding for the light chain of anti-HBsAg monoclonal antibody; KDEL: sequence coding for the KDEL endoplasmic
reticulum retrieval motif; 3´arc: 3´-flanking signal sequence of the arc5-I gene; Pnos: Promoter of the nopaline
synthase gene; nptII: neomycin phosphotransferase II gene; 3´ocs: Terminator of the octopine synthase gene; LB:
T-DNA left border; RB: T-DNA right border
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Online Resource 2 Immunodetection of heavy and light chains of PHB-01 produced in tobacco seeds with goat
anti-mouse HC (Sigma M8770) + rat anti-mouse LC (Sigma K2132) and rabbit anti-goat IgG peroxidase conjugate
(Sigma A5420) + goat anti-rat IgG peroxidase conjugate (Sigma A9037). Lane 1: One-hundred nanograms of PHB01leaf; lanes 2-7: Sixty micrograms of soluble proteins from seed extracts of transgenic lines A10, A35, A50, A68,
A83, A91 respectively; lane 8: Sixty micrograms of soluble proteins from seed extracts of an untransformed plant.
The main degradation products are indicated (*)
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Online Resource 3: Yield and purity of the monoclonal antibody PHB-01seed during purification
Purity (%)c
Yield (%)d
25.0 ± 1.5
78.19 ± 2.26
57.43 ± 4.27
17.1 ± 0.7
93.16 ± 1.13
49.83 ± 3.40
Protein conc.
mAb conc.
(mg/mL) a
(mg/mL) b
Seed extract
6.737 ± 0.133
0.174 ± 0.013
700.4 ± 17.3
Prot. A Elution
3.580 ± 0.178
2.803 ± 0.177
SEC-HPLC/ Conc
3.813 ± 0.119
3.553 ± 0.153
Fraction
Volume (mL)
SEC-HPLC/ Conc: Size exclusion on high performance liquid chromatography TSK G3000 PW and concentration
using 10 kDa cut off devices (Millipore, USA).
a
Determined by BCA.
b
Determined by ELISA.
c
Calculated as the ratio of plantibody concentration determined by ELISA, with respect to the protein
concentration after each purification step X 100.
d
Calculated as the ratio of plantibody yield after each purification step with respect to the plantibody yield in
original seed extract X 100 .
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Online Resource 4: Summary of structural assignments from GU values (glucose units) obtained by profiling the Nglycans from PHB-01seed, released by digestion with PNGase A, on an Amide 80 HPLC column. Representation,
quantification and classification of N-glycans.
Retention
Time
GU expa
GU teob
Structure
58.43
3.32
3.46
59.45
3.39
61.8
Representationc
(%)
Classification
M2
4.5
Oligomannosides
3.46
M2
2.3
Oligomannosides
3.54
-
-
1.0
No identified
71.79
4.23
4.29
F(3)M2
4.6
Complex
(Fucosylated)
74.85
4.48
4.4
M3
4.2
Oligomannosides
80.4
4.92
4.9
M3X
9.9
Complex
(Xylosylated)
81.81
5.04
5.06
A1[3]
2.4
Complex (Axyl/
Afuc)
87.76
5.62
5.5
M3XA1
10.6
Complex
(Xylosylated)
89.17
5.75
5.74
F(3)A1
1.4
Complex
(Fucosylated)
91.31
5.96
5.88
A2X
1.7
Complex
(Xylosylated)
4
a
92.43
6.04
5.9
F(3)M3X
3.3
Complex (Xyl/
Fuc)
93.79
6.23
6.19
M5
1.8
Oligomannosides
94.69
6.34
5.28
F(3)A2
5.6
Complex
(Fucosylated)
96.64
6.56
-
-
1.3
No identified
99.81
6.92
6.85
F(3)A2X
1.8
Complex (Xyl/
Fuc)
101.23
7.09
7.06
M6 D1
6.5
Oligomannosides
107.55
7.92
7.94
M7
29.2
Oligomannosides
113.6
8.83
8.82
M8
5.9
Oligomannosides
117.57
9.48
9.49
M9
1.7
Oligomannosides
Glucose Units, experimental (GU exp): Value calculated from the profile on Amide 80-HPLC of N-glycans from
PHB-01seed released enzymatically by digestion with PNGase, using standard dextran as ladder.
b
Glucose Units, theoretical (GU teo): Values reported in GlycoBase, from the Dublin-Oxford Glycobiology
Laboratory (http://glycobase.ucd.i.e/cgi-bin/profile_upload. cgi).
c
N-glycan representation was performed according to Harvey et al. (2009).
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Online Resource 5 ESI-MS spectrum of underivatized N-glycans from PHB-01seed, released by digestion with Nglycosidase A (PNGase A). All m/z signals correspond to the [M+Na]+ adduct. N-glycan representations follow the
conventions of Harvey et al. (2009). Monosaccharide composition was represented as (Man.GlcNAc.Fuc.Xyl).
GlcNAc: N-acetylglucosamine, Man: mannose, Fuc: fucose, Xyl: xylose
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