Influence of elastin-like polypeptide and hydrophobin on recombinant hemagglutinin accumulations in transgenic tobacco plants

Abstract

Fusion protein strategies are useful tools to enhance expression and to support the development of purification technologies. The capacity of fusion protein strategies to enhance expression was explored in tobacco leaves and seeds. C-terminal fusion of elastin-like polypeptides (ELP) to influenza hemagglutinin under the control of either the constitutive CaMV 35S or the seed-specific USP promoter resulted in increased accumulation in both leaves and seeds compared to the unfused hemagglutinin. The addition of a hydrophobin to the C-terminal end of hemagglutinin did not significantly increase the expression level. We show here that, depending on the target protein, both hydrophobin fusion and ELPylation combined with endoplasmic reticulum (ER) targeting induced protein bodies in leaves as well as in seeds. The N-glycosylation pattern indicated that KDEL sequence-mediated retention of leaf-derived hemagglutinins and hemagglutinin-hydrophobin fusions were not completely retained in the ER. In contrast, hemagglutinin-ELP from leaves contained only the oligomannose form, suggesting complete ER retention. In seeds, ER retention seems to be nearly complete for all three constructs. An easy and scalable purification method for ELPylated proteins using membrane-based inverse transition cycling could be applied to both leaf- and seed-expressed hemagglutinins.

Figure 1. Expression cassettes for hemagglutinin (HA) in plants.

HAs were stably expressed in both leaves and seeds under the control of the CaMV 35S and the seed-specific promoters as the naked form (H5), hydrophobin I fusion protein (H5-HFBI) and ELPylated H5 (H5-ELP). All recombinant HAs contained His and c-myc tags for affinity chromatography purification and Western blotting, respectively. The LeB4 signal peptide and KDEL motif were used to ensure ER retention.

Figure 2. Influence of hydrophobin and elastin-like polypeptide on hemagglutinin accumulation in transgenic plants under the control of either the CaMV 35S promoter (A) or the seed-specific USP promoter (B).

Each column shows the mean value of expression level of the transgenic plants, and the error bars indicate the standard deviation. TSP: total soluble protein.

Figure 3. Expression of influenza HAs in transgenic tobacco plants.

The extracted proteins from transgenic tobacco leaves (A) or seeds (B) were separated by 10% SDS-PAGE, blotted and detected with anti-c-myc monoclonal antibody followed by horseradish peroxidase-linked sheep anti-mouse IgG as a secondary antibody. “+”: anti-hTNFα-VHH-ELP was used as a Western blot standard ; Wt: wild type; TSP: total soluble protein. The numbers refer to independent primary transgenic plants.

Figure 4. Purification of ELPylated hemagglutinin (H5-ELP) from transgenic leaves and seeds by membrane-based ITC.

ELPylated hemagglutinins from leaves (A) and seeds (B) were purified by the standard or improved mITC methods (C) described in the Materials and Methods section. Proteins in the raw plant extract (RE), in the supernatant after passage through a 0.2 µm cellulose acetate membrane (Sm) and in the eluent (Pm) were collected during the mITC purification process and separated by 10% SDS-PAGE. Recombinant proteins were then detected using Coomassie Brilliant Blue staining (left) or an anti-c-myc monoclonal antibody (right).

Figure 5. Purification of non ELPylated HA from transgenic leaves and seeds by His tag-based affinity chromatography.

Non ELPylated HAs from leaves (A) and seeds (B) were purified by the IMAC method described in the materials and methods section. Purified proteins were separated by 10% SDS-PAGE and then detected using Coomassie Brilliant Blue staining (left) or by an anti-c-myc monoclonal antibody (right).

Figure 6. Immunofluorescence analysis of recombinant HAs in plant leaves.

Leaves were fixed, embedded in PEG and sectioned. Recombinant HAs were immunodecorated with an anti-c-myc monoclonal antibody followed by incubation with secondary antibody (anti-mouse-IgG conjugated with AlexaFluor488) and counterstaining with DAPI. A. H5; B. H5-HFBI; C. H5-ELP; D. wild type. Bars represent 50 µm.

Figure 7. Localization of recombinant hemagglutinins in leaves visualized by electron microscopy.

Thin leaf sections mounted on copper grids were probed with monoclonal mouse anti-c-myc antibody followed by the goat anti-mouse-IgG conjugated to 10 nm gold particles. A and B. H5; C. H5-ELP; D. H5-HFBI. The membrane of immunodecorated PB is surrounded by ribosomes (arrows, C). Bars represent 250 nm.

Figure 8. Localization of hemagglutinin-ELP fusions in tobacco seeds.

A, B. Fluorescence microscopy.C, D. Electron microscopy.A, C. Endosperm. B, D. Embryo. Note the ELP bodies (arrowheads, A, B) and those that are loosely packed (arrowheads, C, D). Cell wall (cw), oil bodies (ob), protein storage vacuole (arrows), nucleus (n). Bars 50 µm (A, B), 1 µm (C, D).

Figure 9. Localization of hemagglutinin-hydrophobin I fusions in tobacco seeds.

A. Fluorescence microscopy. B, C. Electron microscopy. B. Endosperm. C. Embryo. Scarce hydrophobin bodies in the endosperm (arrowheads, A, B). Abundant hydrophobin bodies in the embryo cells (arrowheads, A, C). Hydrophobin bodies show non-uniform electron density (*, C). Endosperm (end), embryo (emb), protein storage vacuole (PSV), ribosomes (arrow). Bars 25 µm (A), 0.5 µm (B, C).

Figure 10. Western blot analysis of purified HAs treated/untreated with PNGase F.

Purified HAs from leaves were deglycosylated using the commercial PNGase F enzyme described in the Materials and Methods section. PNGase F-treated and untreated proteins were then separated in 10% SDS-PAGE. Recombinant proteins were detected using an anti-c-myc monoclonal antibody. “−” and “+” indicate PNGase F-untreated and treated samples, respectively.