Figure 1.
Genetic cassette used for plant transformation and evaluation of transgene integration.
(A) Schematic representation of A56 genetic cassette used for tobacco plant transformation. The EPO coding region (red) fused with sequence encoding the TEV protease cleavage site, StrepII tag and KDEL (orange) was placed under the control of a double CaMV 35S promoter, followed by nopaline synthase terminator (nos-T). The human GalT coding region (green) is flanked by a glyceraldehydes-3-phosphate dehydrogease gene (GapC) promoter and terminator. The expression construct has a kanamycin resistance gene neomycin phosphotransferase (nptII) (blue) under the control of nopaline promoter (nos-P). RB/LB, right and left borders. (B) PCR analysis of A56 transgenic lines (lanes 1-12, A56-1 to -12) and CEJ120-12 (CEJ) for the presence of EPO, GalT and nptII in the plant genomic DNA. M, marker; P, plasmid.
Figure 2.
Quantification of asialo-rhuEPO in leaf extracts of transgenic tobacco plants.
(A) A sandwich ELISA was used to determine accumulation levels of asialo-rhuEPO in transgenic plants expressing EPO under the control of CaMV 35S (CEJ120-12) promoter and a 2×35S (A56 1–12) promoter. All data plotted are the average of three independent measurements ± SD. (B) Western blot of total soluble protein (TSP) from CEJ120-12 and selected A56 transgenic lines. (C) Western blot of total leaf protein extracts (TLPE) isolated from transgenic line A56-5. Lanes 1 to 3, 3, 6 and 12 ng of rhuEPOM; lanes 4 and 5, 16 µl (V16) and 32 µl (V32), respectively of A56-5 TLPE. The arrows mark the position of rhuEPOM (lane 1) and asialo-rhuEPOP glycoforms. The expression level of asialo-rhuEPO in leaf tissue was calculated from standard curve generated by measuring the band intensities of known amounts of rhuEPOM and amount of TLPE used. The Western blot analysis was repeated twice. All data plotted are the average of two independent measurements ± SD. Ave, average.
Figure 3.
SDS-PAGE profile (A) and Western blot (B) of purified asialo-rhuEPOP.
(A) Coomassie-stained gel showing commercial rhuEPOM (lane 1, 5 µg) and the asialo-rhuEPOP purified from transgenic line A56-6 (lane 2, 7 µg). M, protein markers. The arrowheads mark the protein bands, which were excised for LC-MS/MS analysis. (B) Western blot of rhuEPOM (lane 1, 5 ng) and purified asialo-rhuEPO (lane 2, 15 ng).
Figure 4.
NSI-FTMS spectrum of PNGase A released and permethylated asialo-rhuEPOP N-glycans.
The schematic glycan structures of the glycans found in N-glycan pool of asialo-rhuEPOP are shown. The structure for each peak was further verified by MS/MS analysis using total ion mapping. The symbols for the glycan structures are: filled blue square, GlcNAc; filled green circle, mannose; filled yellow circle, galactose; filled red triangle, fucose, unfilled star, xylose.
Table 1.
N-Glycans of asialo-rhuEPOP identified by NSI-FTMS and MS/MS analyses.
Figure 5.
Binding profile of asialo-rhuEPOP to ECA-agarose column.
About 800-rhuEPOP (-♦-) or plant-produced rhuEPO lacking both sialic acid and β1,4-galactose (-▴-, negative control) in HEPES-KOH buffer was applied to 1 ml ECA-agarose column. Bound protein was eluted with HEPES-KOH buffer containing 0.2 M lactose. A sandwich ELISA was used to determine the amount of rhuEPO in flow through, wash and eluted fractions. The arrow indicates the start of elution.
Figure 6.
The cytoprotective effect of asialo-rhuEPOP and Western blot of JAK2 and caspase 3.
N2A cells were treated individually with PBS containing 0.1% BSA (vehicle control), 1 µM STS, 1 µM STS+20 U/ml asialo-rhuEPOP (rhuEPOP) or 1 µM STS+20 U/ml rhuEPOM. (A) Cytotoxicity was measured by LDH assay after 12 h treatment. Each experiment had six replicates. All data plotted are the average of three independent experiments ± SD. *, P<0.05. (B) Western blot of activated JAK2 and caspase 3 in cell lysates prepared from cells treated by STS and rhuEPO for 3 and 6 h. For detection of p-JAK2 and JAK2, the blot was probed with anti-p-JAK antibody first followed by stripping the blot and re-probing with anti-total JAK2 antibody. Active caspase 3 was detected using an anti-caspase 3 antibody, which also cross-reacts with procaspase 3. β-actin was used as internal control.