Figure S1: Ma et al., 2015
Supplementary Data 1: Southern blot of three individual plants per generation (T5, T6 and T7), following the digestion of genomic
DNA (10 mg per lane) with NcoI and XbaI. Coding sequences for antibody genes were used as the probe (left panel = heavy chain, right
panel = light chain).
Figure S2: Ma et al., 2015
Supplementary Data 2: Northern blot of individual plants from three generations (three plants from T5, three from T6 and six plants
from T7). Coding sequences for antibody genes were used as probe (left panel = heavy chain transcript, right panel = light chain
transcript). RiboRuler High Range RNA ladder (Fermentas) was used as the MW marker.
Figure S3: Ma et al., 2015
A
Sample
P2G12,
batch 090918
Major signals observed (Da)
148,672
2 x LC + 2 x HC (without CTerm K) +
2 x Hex3HexNAc4FucPent
2 x LC + 1 x HC (without CTerm K) +
1 x HC (with CTerm K)+ 2 x
Hex3HexNAc4FucPent
2 x LC + 2 x HC (without CTerm K) +
2 x Hex3HexNAc4FucPent
2 x LC + 1 x HC (without CTerm K) +
1 x HC (with CTerm K)+ 2 x
Hex3HexNAc4FucPent
148,797
(M-Scan No 1300)
P2G12,
batch 080902
Possible assignment
148,677
148,801
(M-Scan No 1301)
Calculated average
chemical mass (Da)
148,667
148, 795
148,677
148,795
B
Sample
P2G12,
UV peak at RT
(min)
Major signals
observed (Da)
Possible assignments
21.8
23.1
23,322
51,017
1 x LC
1 x HC (without CTerm K) + 1 x
Hex3HexNAc4FucPent
1 x HC + 1 x
Hex3HexNAc4FucPent
1 x LC
1 x HC (without CTerm K) + 1 x
Hex3HexNAc4FucPent
1 x HC + 1 x
Hex3HexNAc4FucPent
batch 090918
(M-Scan No 1300)
23.1
51,145
P2G12,
21.7
22.7
23,321
51,016
batch 080902
(M-Scan No 1301)
22.7
51,144
Calculated
average chemical
mass (Da)
23,325
51,024
51,152
23,325
51,024
51,152
Supplementary Data 3: Calculated chemical mass by LC/ES-MS analysis after A) desalting; and B) reduction.
Figure S4: Ma et al., 2015
Supplementary Data 4: Peptide mapping obtained from online LC/ES-MS analysis of a reduced/carboxymethylated tryptic digest of sample
batches. a) light chain polypeptide; and b) heavy chain polypeptide. Mapped sequences are shown in bold and underlined.
A
B
Figure S5: Ma et al., 2015
Monosaccharide
Fucose
Xylose
Mannose
Galactose
Glucose
GalNAc
GlcNAc
P2G12, clinical batch 090918
Mean nmoles/mg1
Ratio relative to
mannose = 3.02
21
0.9
29
1.3
69
3.0
1.8
0.078
2.3
0.10
ND
/
57
2.5
P2G12, pre-clinical batch 080902
Mean nmoles/mg1
Ratio relative to
mannose = 3.02
16
1.0
22
1.4
48
3.0
1.2
0.075
1.9
0.12
ND
/
39
2.4
Supplementary Data 5: Monosaccharide analysis of P2G12.
Suppl. Data 6: Ma et al., 2015
Figure S6: Map of the plant expression vector pTRAp-2G12-Ds. Open reading frames are shown as arrows. Sequences transferred to
the plant genome are shown in green. (LB/RB – left and right border of T-DNA from A. tumefaciens Ti-plasmid); Pnos – nopaline
synthase gene promoter; pat – Streptomyces hygroscopicus gene for phosphinothricin acetyltransferase; pAnos – nopaline synthase
gene polyadenylation signal; SAR – scaffold attachment region of the N. tabacum RB7 gene; P35SS – CaMV 35S promoter with
duplicated transcriptional enhancer; TL – 5' untranslated region from Tobacco etch virus; pA35S – CaMV 35S polyadenylation signal;
2G12Hc – 2G12 heavy chain gene, including native signal peptide (SPg); 2G12LC - 2G12 light chain gene, including native signal peptide
(SPk); TP Hordeum vulgare GBSSI transit peptide sequence; DsRed – Discosoma spp. red fluorescent protein gene; RK2 ori – origin of
replication; bla – b-lactamase gene; ColE1ori – origin of replication.