A recombinant multimeric immunoglobulin expressed in rice shows assembly-dependent subcellular localization in endosperm cells

To investigate the role of subunit assembly in the intracellular deposition of multimeric recombinant proteins, we expressed a partially humanized secretory immunoglobulin in rice endosperm cells and determined the subcellular locations of the assembled protein and its individual components. Transgenic rice plants expressing either individual subunits or all the subunits of the antibody were generated by particle bombardment, and protein localization was determined by immunoelectron microscopy. Assembly of the antibody was confirmed by immunoassay and coimmunoprecipitation. Immunolocalization experiments showed no evidence for secretion of the antibody or any of its components to the apoplast. Rather, the nonassembled light chain, heavy chain and secretory component accumulated predominantly within endoplasmic reticulum-derived protein bodies, while the assembled antibody, with antigen-binding function, accumulated specifically in protein storage vacuoles. These results show that the destination of a complex recombinant protein within the plant cell is influenced by its state of assembly.     

Gluten quality of bread wheat is associated with activity of RabD GTPases

In the developing endosperm of bread wheat (Triticum aestivum), seed storage proteins are produced on the rough endoplasmic reticulum (ER) and transported to protein bodies, specialized vacuoles for the storage of protein. The functionally important gluten proteins of wheat are transported by two distinct routes to the protein bodies where they are stored: vesicles that bud directly off the ER and transport through the Golgi. However, little is known about the processing of glutenin and gliadin proteins during these steps or the possible impact on their properties. In plants, the RabD GTPases mediate ER‐to‐Golgi vesicle transport. Available sequence information for Rab GTPases in Arabidopsis, rice, Brachypodium and bread wheat was compiled and compared to identify wheat RabD orthologs. Partial genetic sequences were assembled using the first draft of the Chinese Spring wheat genome. A suitable candidate gene from the RabD clade (TaRabD2a) was chosen for down‐regulation by RNA interference (RNAi), and an RNAi construct was used to transform wheat plants. All four available RabD genes were shown by qRT‐PCR to be down‐regulated in the transgenic developing endosperm. The transgenic grain was found to produce flour with significantly altered processing properties when measured by farinograph and extensograph. SE‐HPLC found that a smaller proportion of HMW‐GS and large proportion of LMW‐GS are incorporated into the glutenin macropolymer in the transgenic dough. Lower protein content but a similar protein profile on SDS‐PAGE was seen in the transgenic grain.     

Rice endosperm is cost‐effective for the production of recombinant griffithsin with potent activity against HIV

Protein microbicides containing neutralizing antibodies and antiviral lectins may help to reduce the rate of infection with human immunodeficiency virus (HIV) if it is possible to manufacture the components in large quantities at a cost affordable in HIV‐endemic regions such as sub‐Saharan Africa. We expressed the antiviral lectin griffithsin (GRFT), which shows potent neutralizing activity against HIV, in the endosperm of transgenic rice plants (Oryza sativa), to determine whether rice can be used to produce inexpensive GRFT as a microbicide ingredient. The yield of ^OSGRFT in the best‐performing plants was 223 μg/g dry seed weight. We also established a one‐step purification protocol, achieving a recovery of 74% and a purity of 80%, which potentially could be developed into a larger‐scale process to facilitate inexpensive downstream processing. ^OSGRFT bound to HIV glycans with similar efficiency to GRFT produced in Escherichia coli. Whole‐cell assays using purified ^OSGRFT and infectivity assays using crude extracts of transgenic rice endosperm confirmed that both crude and pure ^OSGRFT showed potent activity against HIV and the crude extracts were not toxic towards human cell lines, suggesting they could be administered as a microbicide with only minimal processing. A freedom‐to‐operate analysis confirmed that GRFT produced in rice is suitable for commercial development, and an economic evaluation suggested that 1.8 kg/ha of pure GRFT could be produced from rice seeds. Our data therefore indicate that rice could be developed as an inexpensive production platform for GRFT as a microbicide component.     

Influence of elastin-like peptide fusions on the quantity and quality of a tobacco-derived human immunodeficiency virus-neutralizing antibody

The use of vaginal microbicides containing human immunodeficiency virus (HIV)-neutralizing antibodies (nAbs) is a promising strategy to prevent HIV-1 infection. Although antibodies are predominantly manufactured using mammalian cells, elastin-like peptide (ELP) fusion technology improves the stability of recombinant, plant-produced proteins and facilitates their purification, making plants an alternative platform for antibody production. We generated transgenic tobacco plants accumulating four different formats of the anti-HIV-1 antibody 2G12 in the endoplasmic reticulum (ER), i.e. with ELP on either the light or heavy chain, on both, or on neither. Detailed analysis of affinity-purified antibodies by surface plasmon resonance spectroscopy showed that the kinetic binding parameters of all formats were identical to 2G12 lacking ELP produced in Chinese hamster ovary (CHO) cells. Importantly, protein purification from seeds by inverse transition cycling (ITC) did not affect the binding kinetics. Analysis of heavy chain N-glycans from leaf-derived antibodies showed that retrieval to the ER was efficient for all formats. In seeds, however, N-glycans on the naked antibody were extensively trimmed compared with those on the ELP fusion formats, and were localized to a different subcellular compartment. The in vitro HIV-neutralization properties of the tobacco-derived 2G12 were equivalent to or better than those of the CHO counterpart.     

Proteomic analysis of rice endosperm cells in response to expression of hGM-CSF

The accumulation of significant levels of transgenic products in plant cells is required not only for crop improvement, but also for molecular pharming. However, knowledge about the fate of transgenic products and endogenous proteins in grain cells is lacking. Here, we utilized a quantitative mass spectrometry-based proteomic approach for comparative analysis of expression profiles of transgenic rice endosperm cells in response to expression of a recombinant pharmaceutical protein, human granulocyte-macrophage colony stimulation factor (hGM-CSF). This study provided the first available evidence concerning the fate of exogenous and endogenous proteins in grain cells. Among 1883 identified proteins with a false positive rate of 5%, 103 displayed significant changes (p-value < 0.05) between the transgenic and the wild-type endosperm cells. Notably, endogenous storage proteins and most carbohydrate metabolism-related proteins were down-regulated, while 26S proteasome-related proteins and chaperones were up-regulated in the transgenic rice endosperm. Furthermore, it was observed that expression of hGM-CSF induced endoplasmic reticulum stress and activated the ubiquitin/26S-proteasome pathway, which led to ubiquitination of this foreign gene product in the transgenic rice endosperm.     

Production of an anti-mouse MHC class II monoclonal antibody with biological activity in transgenic tobacco

To produce a monoclonal antibody specific to a mouse major histocompatibility complex (MHC) class II protein, we synthesized the complementary DNAs for the heavy and light chains of a monoclonal antibody by PCR amplification. These cDNAs were then introduced separately into tobacco plant cells. After performing Northern blot analysis to confirm the expression of each of the chain genes in the transformed plants, we constructed transgenic plants expressing both the heavy and light chains by sexual crossing. The expression of the heavy and light chain genes in the sexually crossed plant was confirmed by Northern and Western blot analyses, respectively. Fluorocytometric analysis showed that the plant-derived antibodies, which we purified using a protein G affinity column, bound specifically to target cells that expressed the cognate MHC class II molecules on their cell surfaces. The results of this study demonstrate that a monoclonal antibody against mouse MHC class II proteins can be expressed in transgenic plants. They also show the specific binding activity of plant-derived antibodies to cognate antigens.     

The endoplasmic reticulum stress induced by highly expressed OsrAAT reduces seed size via pre-mature programmed cell death

The high accumulation of a recombinant protein in rice endosperm causes endoplasmic reticulum (ER) stress and in turn dramatically affects endogenous storage protein expression, protein body morphology and seed phenotype. To elucidate the molecular mechanisms underlying these changes in transgenic rice seeds, we analyzed the expression profiles of endogenous storage proteins, ER stress-related and programmed cell death (PCD)-related genes in transgenic lines with different levels of Oryza sativa recombinant alpha antitrypsin (OsrAAT) expression. The results indicated that OsrAAT expression induced the ER stress and that the strength of the ER stress was dependent on OsrAAT expression levels. It in turn induced upregulation of the expression of the ER stress response genes and downregulation of the expression of the endogenous storage protein genes in rice endosperm. Further experiments showed that the ER stress response upregulated the expression of PCD-related genes to disturb the rice endosperm development and induced pre-mature PCD. As consequence, it resulted in decrease of grain weight and size. The mechanisms for the detriment seed phenotype in transgenic lines with high accumulation of the recombinant protein were elucidated.     

Rapid High-Level Production of Functional HIV Broadly Neutralizing Monoclonal Antibodies in Transient Plant Expression Systems

Passive immunotherapy using anti-HIV broadly neutralizing monoclonal antibodies (mAbs) has shown promise as an HIV treatment, reducing mother-to-child-transmission (MTCT) of simian/human immunodeficiency virus (SHIV) in non-human primates and decreasing viral rebound in patients who ceased receiving anti-viral drugs. In addition, a cocktail of potent mAbs may be useful as mucosal microbicides and provide an effective therapy for post-exposure prophylaxis. However, even highly neutralizing HIV mAbs used today may lose their effectiveness if resistance occurs, requiring the rapid production of new or engineered mAbs on an ongoing basis in order to counteract the viral resistance or the spread of a certain HIV-1 clade in a particular region or patient. Plant-based expression systems are fast, inexpensive and scalable and are becoming increasingly popular for the production of proteins and monoclonal antibodies. In the present study, Agrobacterium-mediated transient transfection of plants, utilizing two species of Nicotiana, have been tested to rapidly produce high levels of an HIV 89.6PΔ140env and several well-studied anti-HIV neutralizing monoclonal antibodies (b12, 2G12, 2F5, 4E10, m43, VRC01) or a single chain antibody construct (m9), for evaluation in cell-based viral inhibition assays. The protein-A purified plant-derived antibodies were intact, efficiently bound HIV envelope, and were equivalent to, or in one case better than, their counterparts produced in mammalian CHO or HEK-293 cells in both neutralization and antibody dependent viral inhibition assays. These data indicate that transient plant-based transient expression systems are very adaptable and could rapidly generate high levels of newly identified functional recombinant HIV neutralizing antibodies when required. In addition, they warrant detailed cost-benefit analysis of prolonged incubation in plants to further increase mAb production.     

Characterization of VRC01, a potent and broadly neutralizing anti‐HIV mAb,produced in transiently and stably transformed tobacco

The proposed clinical trial in Africa of VRC01, a potent broadly neutralizing antibody (bNAb) capable of neutralizing 91% of known HIV‐1 isolates, raises concerns about testing a treatment which will be too expensive to be accessible by the most important target population, the poor in under‐developed regions such as sub‐Saharan Africa. Here, we report the expression of VRC01 in plants as an economic alternative to conventional mammalian‐cell‐based production platforms. The heavy and light chain genes of VRC01 were cloned onto a single vector, pTRAk.2, which was transformed into Nicotiana benthamiana or Nicotiana tabacum using transient and stable expression production systems respectively. VRC01 has been successfully expressed transiently in plants with expression level of approximately 80 mg antibody/kg; stable transgenic lines expressing up to 100 mg antibody/kg were also obtained. Plant‐produced VRC01 from both systems showed a largely homogeneous N‐glycosylation profile with a single dominant glycoform. The binding kinetics to gp120 IIIB (approximately 1 nm ), neutralization of HIV‐1 BaL or a panel of 10 VRC01‐sensitive HIV‐1 Env pseudoviruses of VRC01 produced in transient and stable plants were also consistent with VRC01 from HEK cells.     

Antisense suppression of LOX3 gene expression in rice endosperm enhances seed longevity

Lipid peroxidation plays a major role in seed longevity and viability. In rice grains, lipid peroxidation is catalyzed by the enzyme lipoxygenase 3 (LOX3). Previous reports showed that grain from the rice variety DawDam in which the LOX3 gene was deleted had less stale flavour after grain storage than normal rice. The molecular mechanism by which LOX3 expression is regulated during endosperm development remains unclear. In this study, we expressed a LOX3 antisense construct in transgenic rice (Oryza sativa L.) plants to down‐regulate LOX3 expression in rice endosperm. The transgenic plants exhibited a marked decrease in LOX mRNA levels, normal phenotypes and a normal life cycle. We showed that LOX3 activity and its ability to produce 9‐hydroperoxyoctadecadienoic acid (9‐HPOD) from linoleic acid were significantly lower in transgenic seeds than in wild‐type seeds by measuring the ultraviolet absorption of 9‐HPOD at 234 nm and by high‐performance liquid chromatography. The suppression of LOX3 expression in rice endosperm increased grain storability. The germination rate of TS‐91 (antisense LOX3 transgenic line) was much higher than the WT (29% higher after artificial ageing for 21 days, and 40% higher after natural ageing for 12 months). To our knowledge, this is the first report to demonstrate that decreased LOX3 expression can preserve rice grain quality during storage with no impact on grain yield, suggesting potential applications in agricultural production.     

Biochemical and functional characterization of anti-HIV antibody-ELP fusion proteins from transgenic plants

The stability and recovery of recombinant proteins expressed in plants are improved by fusion to elastin-like peptides (ELPs). In order to test the suitability of ELP for the production of pharmaceutical proteins, transgenic plants were created that individually expressed the light and heavy chains of the broadly neutralizing anti-human immunodeficiency virus type 1 (anti-HIV-1) monoclonal antibody 2F5, which is being evaluated as a microbicide component. The antibody chains were expressed both with and without a C-terminal ELP fusion. Crossing these plants in all combinations resulted in transgenic lines producing the full antibody in four formats, with ELP on either the light or heavy chains, on both or on neither. Characterization of the affinity-purified antibodies by surface plasmon resonance spectroscopy showed that the kinetic binding parameters were identical to those of a Chinese hamster ovary (CHO) cell counterpart lacking ELP. N -Glycan analysis showed that all four derivatives contained predominantly oligo-mannose-type N -glycans and that the ELP fusions had no significant effect on N -glycan structure. It was concluded that ELP fusion to the light chain, heavy chain or both chains of a plant-derived antibody had no adverse affects on protein quality, but had a positive impact on the yield. ELP fusions do not interfere with folding, assembly, trafficking in the secretory pathway or post-translational modification, but enhance stability whilst at the same time simplifying recovery.     

Monoclonal antibody engineering in plants.

Techniques for plant transformation have been developed to such an extent that a number of foreign genes are currently being introduced into transgenic plants. Tobacco plants that produce monoclonal antibodies are of interest, because in addition to synthesis of two gene products (i.e. the heavy and light chains), the two polypeptides need to be assembled correctly, in order to result in a functional antibody. The studies on a catalytic antibody suggest that this is the case, and that the antibody functions identically to the native murine-derived antibody. The only difference observed was in the glycosylation of the heavy chain. Further transgenic plants are being generated to produce monoclonal antibodies that may be used therapeutically (and are therefore required in large quantities), or to provide disease resistance in plants. In addition, the ability of plants to assemble antibody complexes is being investigated further, to study the possibility of generating secretory IgA, which consists of heavy and light chains as well as two additional polypeptide units.     

Sorting of Glycoprotein B from Human Cytomegalovirus to Protein Storage Vesicles in Seeds of Transgenic Tobacco

As part of ongoing studies into the use of plant expression systems for making human therapeutic proteins, we have successfully expressed the major glycoprotein, gB, of human cytomegalovirus (HCMV) in transgenic tobacco plants. Viral glycoprotein was detectable in the protein extracts of mature tobacco seeds using neutralizing and non-neutralizing monoclonal antibodies specific for gB. Although several mammalian proteins have been expressed in tobacco, localization of these proteins in transgenic tobacco tissue has not been extensively examined. The objective of this study was to identify the site(s) of recombinant gB deposition in mature tobacco seeds. Using immunogold labelling and electron microscopy, we found specific labelling for gB in the endosperm of transgenic seeds, with gB localized almost exclusively in protein storage vesicles (PSV). This occurred in seeds that were freshly harvested and in seeds that had been stored for several months. These data indicate that gB behaves like a plant storage protein when expressed in tobacco seeds, and provide further support for the suitability of plants for producing recombinant proteins of potential clinical relevance.     

Expression and localization of human lysozyme in the endosperm of transgenic rice

In order to understand the characteristics of recombinant protein expression and sublocalization in rice ( Oryza sativa L.) endosperm, we examined the expression level of human lysozyme protein and its subcellular location in transgenic rice seeds driven by rice glutelin and globulin promoters and signal peptides. A time course of human lysozyme expression during endosperm development was analyzed. The results showed that the expression profile of recombinant protein accumulation in endosperm paralleled that of the two storage proteins. Immunofluorescence microscopy revealed that human lysozyme and storage proteins co-localized to type-II protein bodies. Both promoter-signal peptide parings targeted recombinant protein to the protein bodies. In addition, a transgenic line with a higher lysozyme expression level exhibited morphologically different protein bodies with an unbalanced composition of lysozyme and native storage proteins. The high-level expression of recombinant protein distorted the trafficking and sorting of native storage proteins in rice endosperm and affected the expression of native storage protein.     

Site‐specific proteolytic degradation of IgG monoclonal antibodies expressed in tobacco plants

Plants are promising hosts for the production of monoclonal antibodies (mAbs). However, proteolytic degradation of antibodies produced both in stable transgenic plants and using transient expression systems is still a major issue for efficient high‐yield recombinant protein accumulation. In this work, we have performed a detailed study of the degradation profiles of two human IgG1 mAbs produced in plants: an anti‐HIV mAb 2G12 and a tumour‐targeting mAb H10. Even though they use different light chains (κ and λ, respectively), the fragmentation pattern of both antibodies was similar. The majority of Ig fragments result from proteolytic degradation, but there are only a limited number of plant proteolytic cleavage events in the immunoglobulin light and heavy chains. All of the cleavage sites identified were in the proximity of interdomain regions and occurred at each interdomain site, with the exception of the V_L/C_L interface in mAb H10 λ light chain. Cleavage site sequences were analysed, and residue patterns characteristic of proteolytic enzymes substrates were identified. The results of this work help to define common degradation events in plant‐produced mAbs and raise the possibility of predicting antibody degradation patterns ‘a priori’ and designing novel stabilization strategies by site‐specific mutagenesis.     

Accumulation of Soybean Glycinin and Its Assembly with the Glutelins in Rice

Saline-soluble glycinins and insoluble glutelins are the major storage proteins in soybean (Glycine max) and rice (Oryza sativa), respectively. In spite of their differences in solubility properties, both proteins are members of the 11S globulin gene family based on their similarities in primary sequences and processing of the coded protein. Wild-type and methionine-modified glycinin coding sequences were expressed in transgenic rice plants under the control of the rice glutelin GluB-1 promoter. Glycinins were specifically synthesized in the endosperm tissue and co-localized with glutelins in type II protein bodies. They assembled into 7S and 11S species, similar to what was observed in developing soybean seeds. This pattern was quite different from that displayed by the rice glutelins in untransformed plants, in which processed subunits sedimenting at 2S were apparent. In glycinin-expressing transgenic plants, however, glutelins were observed sedimenting at 7S and 11S with lesser amounts in the 2S region. A portion of the glycinins was also found associated in the insoluble glutelin fraction. Renaturation experiments suggested that the hybrid glycinin-glutelin oligomers were formed through specific interactions. Overall, these results indicate that despite significant differences in the assembly of soybean glycinin and rice glutelin, both proteins can assemble with each other to form soluble hexameric oligomers or insoluble aggregates.     

Production of monoclonal antibodies with a controlled N‐glycosylation pattern in seeds of Arabidopsis thaliana

Seed‐specific expression is an appealing alternative technology for the production of recombinant proteins in transgenic plants. Whereas attractive yields of recombinant proteins have been achieved by this method, little attention has been paid to the intracellular deposition and the quality of such products. Here, we demonstrate a comparative study of two antiviral monoclonal antibodies (mAbs) (HA78 against Hepatitis A virus; 2G12 against HIV) expressed in seeds of Arabidopsis wild‐type (wt) plants and glycosylation mutants lacking plant specific N‐glycan residues. We demonstrate that 2G12 is produced with complex N‐glycans at great uniformity in the wt as well as in the glycosylation mutant, carrying a single dominant glycosylation species, GnGnXF and GnGn, respectively. HA78 in contrast, contains additionally to complex N‐glycans significant amounts of oligo‐mannosidic structures, which are typical for endoplasmic reticulum (ER)‐retained proteins. A detailed subcellular localization study demonstrated the deposition of both antibodies virtually exclusively in the extracellular space, illustrating their efficient secretion. In addition, although a KDEL‐tagged version of 2G12 exhibited an ER‐typical N‐glycosylation pattern, it was surprisingly detected in protein storage vacuoles. The different antibody variants showed different levels of degradation with hardly any degradation products detectable for HA78 carrying GnGnXF glycans. Finally, we demonstrate functional integrity of the HA78 and 2G12 glycoforms using viral inhibition assays. Our data therefore demonstrate the usability of transgenic seeds for the generation of mAbs with a controlled N‐glycosylation pattern, thus expanding the possibilities for the production of optimally glycosylated proteins with enhanced biological activities for the use as human therapeutics.     

Targeted transcriptomic and metabolic profiling reveals temporal bottlenecks in the maize carotenoid pathway that may be addressed by multigene engineering

Carotenoids are a diverse group of tetraterpenoid pigments found in plants, fungi, bacteria and some animals. They play vital roles in plants and provide important health benefits to mammals, including humans. We previously reported the creation of a diverse population of transgenic maize plants expressing various carotenogenic gene combinations and exhibiting distinct metabolic phenotypes. Here we performed an in‐depth targeted mRNA and metabolomic analysis of the pathway to characterize the specific impact of five carotenogenic transgenes and their interactions with 12 endogenous genes in four transgenic lines representing distinct genotypes and phenotypes. We reconstructed the temporal profile of the carotenoid pathway during endosperm development at the mRNA and metabolic levels (for total and individual carotenoids), and investigated the impact of transgene expression on the endogenous pathway. These studies enabled us to investigate the extent of any interactions between the introduced transgenic and native partial carotenoid pathways during maize endosperm development. Importantly, we developed a theoretical model that explains these interactions, and our results suggest genetic intervention points that may allow the maize endosperm carotenoid pathway to be engineered in a more effective and predictable manner.