Figure 1.
Schematic presentation of expression vectors SP and SA.
The expression cassettes for PAL protein, ELP-intein-PAL fusion protein and hygromycin transferase were presented. RB, right border; LB, left border; Gt1 pro, rice glutelin GluA promoter; 35S pro, CaMV 35S promoter; LN, linker sequence; HYG, hygromycin phosphotransferase gene; NOSter, NOS terminator.
Figure 2.
Expression of PAL in transgenic rice seeds.
(A) Analysis of total protein extracted from transgenic rice seeds by SDS-PAGE and by immunoblot using anti-PAL antibody, anti-PAL. WT, non-trangenic rice; and M, Precision plus protein Standards (Bio-Rad). Empty trangle denoted the band of synthesized EiP in SA seeds and black triangle denoted the immunoactive band of N-glycosylated PAL. Arrows denoted PAL and ELP-intein-PAL fusion protein (EiP). (B) N-Glycosylation analysis of PAL in SP (top panel) and EiP in SA seeds (bottom panel) by Endo H digestion. gPAL, N-glycosylated PAL and gEiP, N-glycosylated EiP. (C) Detection of Ei tag in total protein extracted from SA seeds by immunoblot using anti-ELP antibody. Only EiP fusion protein was detected while free Ei tag was not observed. Arrows denoted N-glycosylated PAL (gPAL), PAL and ELP-intein-PAL fusion protein (EiP). (D) Immunoblot analysis on total protein extracted from SA seeds of different trnagenic lines (1–6) using anti-PAL antibody. (E) Immunoblot analysis of recombinant proteins extraction from transgenic rice seeds by four different extraction buffers, B1, B2, B3 and To. Total protein samples extracted from equal amount of rice seed powder by different buffers (see Materials and Methods) were used for analysis. (F) Relative accumulation levels of PAL in SP and SA rice seeds. gPAL, N-glycosylated PAL; and ePAL, relative amount of PAL derived from ELP-intein-PAL fusion protein. (G) Accumulation levels of uncleaved EiP in T1 and T2 seeds of SA transformed rice. Error bars indicate standard deviation among different transgenic lines.
Figure 3.
Subcellular localization of PAL in endosperm cells of transgenic rice seeds.
(A–B) Immunogold labeling of WT (A) and SP (B) endosperm cells using anti-PAL antibody. Gold particles were indicated by arrows. (C–D) Immunogold labeling of SA endosperm cells using anti-PAL antibody (C) and with anti-ELP antibody (D). (E) Novel irregular protein bodies, iPBs, as indicated by *, were formed in endosperm cells of SA seeds. (F) The iPBs at the protein body-ER site were also observed. Arrows indicated the labeled gold particles while arrowheads ribosomes on rough ER membrane. PSV, protein storage vacuoles; CW, cell wall; PB-I, type I protein bodies; and bar, 500 nm.
Figure 4.
Co-localization of EiP and prolamins in the irregular protein bodies.
(A–C) Prolamins labeling with FITC fluorescence in WT (A), SP (B) and SA (C) endosperm cells. (D) ELP labeling with Rhodamine Red fluorescence in SA endosperm cells. (E) Merged pictures of (C) and (D) Bars, 10 µm. (F) Immuno-gold labeling with anti-prolamin antibody confirmed the deposition of prolamins in the iPBs (indicated by arrows). Bar, 500 nm.
Figure 5.
Schematic illustration of the purification of ELP-intein fusion protein from rice seeds.
Figure 6.
Purification of recombinant PAL from transgenic rice seeds by the ELP-intein system.
The whole purification was targeted at EiP fusion protein without considering the in vivo cleaved PAL. (A) Analysis of total protein by SDS-PAGE and by immunoblot using anti-ELP antibody, anti-ELP. Equal amount of total protein was loaded in all lanes. To, extracted by total protein extraction buffer; Bt, extracted by Bt buffer. (B) Relative purification efficiency of EiP from transgenic rice seeds. Samples EX, EXF, DS, DSC, 1CS, 2CS, 3CS, FP and FS, as denoted in the Purification Scheme and Materials and Methods. Recovery percentage of EiP was counted relative to EiP extracted by total protein extraction buffer, and error bar was obtained from three independent experiments. (C–D) Purification of PAL from rice seeds by the ELP-intein system: (C) SDS-PAGE analysis; (D) immunoblot reacted with anti-ELP antibody, anti-ELP and anti-PAL antibody, anti-PAL. Lanes EX, EXF, DS, DSC, 1CS, 2CS, 3CS, FP and FS, as denoted in the Purification Scheme and Materials and Methods; and M, Precision Plus protein Standards (Bio-Rad). Arrows indicated ELP-intein-PAL fusion protein (EiP), cleaved ELP-intein tag (Ei), and purified PAL and N-glycosylated PAL (gPAL).