Given the advantages discussed above, maize is favored among the cereals as a platform for the large-scale production of antibodies such as the HIV neutralizing antibody 2G12 that has been successfully produced in maize seeds, reaching yields of 75 µg/g [2, 8]. 2G12 antibody recognizes oligomanose-type glycans on the gp120 envelope protein of human immunodeficiency virus 1 (HIV-1) and is considered a promising microbiocide .
Maize seeds accumulate proteins both in protein storage vacuoles and in protein bodies that directly derive from the endoplasmic reticulum. Maize prolamins (α-, γ- and δ-zeins) assemble in protein bodies which comprise a central core of α- and δ-zeins and a peripherical layer of γ-zeins . The zeins interact non-covalently  but are also cross-linked by intermolecular disulfide bridges, particularly the γ-zeins which are cysteine rich and form covalently-linked multimeric aggregates that initiate the formation of protein bodies. This ability of formation of disulfide bridges, together with the abundance of zeins in maize endosperm (up to 50 % of total protein in mature seeds ) makes it likely that the presence of recombinant proteins such as antibodies, that also form intermolecular disulfide bridges would induce the formation of oligomers. Antibody chain covalently linked to γ-zein would not be reflected in the yield of 2G12 antibody previously reported following saline extraction . We suggest that the potential of maize as an expression platform for soluble recombinant antibody could be boosted in maize varieties with lower levels of endogenous γ-zein.
Material and methods
Transgenic maize seeds expressing 2G12 carrying a signal peptide that directs the antibody chains into the endomembrane system but no further targeting signal were fixed in 4 % paraformaldehyde and 0,5 % glutaraldehyde. Small pieces of developing transgenic seeds (~1 mm3) were further processed as described before . After dehydration through ethanol series, samples were infiltrated and embedded in LRWhite resin and the blocks were allowed to polymerise at 60 °C for 2 days. Semithin sections (1 µm) were obtained with a Leica Ultracut UCT and stained with toluidine blue or used for immunofluorescence studies. Ultrathin sections (80 nm) were also obtained and stained with uranyl acetate 1 % and lead citrate 3 % in a Leica EM AC20 after immunogold localization of recombinant 2G12 antibody. Light and immunofluorescence pictures were taken with a Leica DM5500. Ultrathin sections were observed with a FEI Tecnai G2 electron microscope.
Results and discussion
The deposition of the antibody 2G12 in maize endosperm was studied. Maize endosperm cells are characterized by the presence of abundant spheroidal starch grains, small spherical zein bodies spread evenly within the cytoplasma and several protein storage vacuoles (PSV) (Figure 1A). Antibody chains were localized within such PSVs (Figure 1B), as expected for a protein entering the endomembrane system in cereal seeds with no further targeting information [14, 15]. Unexpectedly, a strong signal could be also detected within the zein bodies, contrary to what has been reported for other secretory recombinant proteins expressed in maize .
Fig. 1: Deposition of 2G12 antibody in maize endosperm cells. A) Light microscopy, toluidine blue. See the protein storage compartments in maize endosperm cells, protein storage vacuoles (arrows) and zein bodies (arrowheads). B) Fluorescence microscopy. Strong labelling in the protein storage vacuoles (arrow) and within the zein bodies (arrowheads). Nucleus (n), starch (s). Bars 20 µm.
The electron microscope confirmed this deposition and, moreover, showed that some zein bodies had lost their typical spherical appearance and the normal distribution of zeins with the protein body (Figure 2). Lending and Larkins  described zein bodies as spherical structures with a low electrondense core of α- and δ-zeins and a thin, high electrondense layer of γ-zeins in the periphery. This structure is conserved and it has been shown that the presence and balanced interactions of all zein subfamilies is necessary to stabilize the zein bodies and form typical spherical structures [16, 17, 18]. In our samples, a significant fraction of the antibody could only be extracted under reducing conditions, pointing at the formation of disulphide bridges between antibody chains and γ-zeins . This interaction is also supported by the fact that the prevalence of the amorphous zein bodies was dependent of the amount of 2G12 expressed, indicating that the presence of zein-antibody oligomers was the responsible of the malformations observed in the zein bodies . While in some cases interaction of recombinant pharmaceuticals with endogenous proteins are desired aiming at oral delivery, in others, like the present case, they affect the yield of soluble protein. We suggest that minimizing interaction with endogenous storage proteins should be a valid strategy to enhance the production of soluble recombinant antibodies in maize.
Fig. 2: Zein bodies in WT seeds (A) and in transgenic seeds (B). Electronmicroscopy, general non-specific staining. WT zein bodies (zb) are spherical and have a low electrondense central core, surrounded by a thin layer of high electrondensity. Some zein bodies in the transgenic seeds are amorphous and show a discontinuous electrondense layer on the periphery (zb*). Bars 0,5 µm.
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