Generation and Assembly of Secretory Antibodies in Plants

K.-C., J.; Hiatt, Andrew; Hein, Mich B.; Vine, Nicholas D.; Wang, Fei; Stabila, P.; Dolleweerd, Craig J. van; Mostov, Keith E.; Lehner, Thomas

doi:10.1126/science.7732380

Cited by 540 publications

(268 citation statements)

References 24 publications

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“…Ma and co-workers demonstrated that plant cells can assemble mammalian antibodies in a secretory form: tobacco plants transformed with all four genes encoding chimeric sIgA/G produced fully functional secretory antibodies [34].…”

Section: Deleted: Itsmentioning

confidence: 99%

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Manufacturing antibodies in the plant cell

Orzáez

Granell

Blázquez

2009

Biotechnology Journal

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International audiencePlants have long been considered advantageous platforms for large-scale production of antibodies due to their low cost, scalability, and the low chances of pathogen contamination. Much effort has therefore been devoted to efficiently produce mAbs (from nanobodies to secretory antibodies) in plant cells. Several technical difficulties have been encountered and are being coped with. Improvements in production levels have been achieved by manipulation of gene expression and, more efficiently, of cell targeting and protein folding and assembly. Differences in mAb glycosylation patterns between animal and plant cells are being successfully addressed by the elimination and introduction of the appropriate enzyme activities in plant cells. Another relevant battlefield is the dichotomy between production capacity and speed. Classically, stably transformed plant lines have been proposed for large scale mAb production, whereas the use of transient expression systems has always provided production speed at the cost of scalability. However, recent advances in transient expression techniques have brought impressive yield improvements, turning speed and scalability highly compatible assets. In the era of personalized medicines, the combination of yield and speed, and the advances in glyco-engineering have made the plant cell a serious contender in the field of recombinant antibody production

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Section: Deleted: Itsmentioning

confidence: 99%

“…Deleted: [20,34] Deleted: [35] Deleted: SIgA In their natural biofactories, the lymphocytes, antibodies are synthesized through the cell endomembrane system. Antibody synthesis requires coordinated expression, folding and assembly of H and L chains.…”

Section: Deleted: Alsomentioning

confidence: 99%

Manufacturing antibodies in the plant cell

Orzáez

Granell

Blázquez

2009

Biotechnology Journal

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“…To express the polyhydroxybutyrate polymer or Guy's 13 antibody, for example, single genes were first introduced into individual transgenic plants, then these plants were back-crossed to reconstitute the entire pathway or the complete protein 2,3 . Similarly, in a seven year long effort, Ye et al 4 recently introduced a set of three genes for a short biosynthetic pathway that resulted in β-carotene expression in rice.…”

Section: Abstract Polycistrons; Plastid Transformation; Gm Crops; Bt mentioning

confidence: 99%

Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals

et al. 2001

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In nuclear transgenic plants, expression of multiple genes requires introduction of individual genes and time-consuming subsequent backcrosses to reconstitute multi-subunit proteins or pathways, a problem that is compounded by variable expression levels. In order to accomplish expression of multiple genes in a single transformation event, we have introduced several genes into the chromoplast genome. We confirmed stable integration of the cry2Aa2 operon by PCR and Southern blot analyses in T 0 and T 1 transgenic plants. Foreign protein accumulated at 45.3% of the total soluble protein in mature leaves and remained stable even in old bleached leaves (46.1%), thereby increasing the efficacy and safety of transgenic plants throughout the growing season. This represents the highest level of foreign gene expression reported in transgenic plants to date. Insects that are normally difficult to control (10-day old cotton bollworm, beet armyworm) were killed 100% after consuming transgenic leaves. Electron micrographs showed the presence of the insecticidal protein folded into cuboidal crystals. Formation of crystals of foreign proteins (due to hyperexpression and folding by the putative chaperonin, ORF 2) provides a simple method of purification by centrifugation and enhances stability by protection from cellular proteases. Demonstration of expression of an operon in transgenic plants paves the way to engineering new pathways in plants in a single transformation event. Keywords polycistrons; plastid transformation; GM crops; Bt resistanceIn plant and animal cells, the monocistronic translation of nuclear messenger RNAs (mRNAs) poses problems in engineering multiple genes in plants 1 . To express the polyhydroxybutyrate polymer or Guy's 13 antibody, for example, single genes were first introduced into individual transgenic plants, then these plants were back-crossed to reconstitute the entire pathway or the complete protein 2,3 . Similarly, in a seven year long effort, Ye et al. 4 recently introduced a set of three genes for a short biosynthetic pathway that resulted in β-carotene expression in rice. In contrast, most chloroplast genes of higher * Corresponding author: daniell@mail.ucf.edu. HHS Public Access Results Chloroplast vectorThe 4.0 kb cry2Aa2 operon was inserted into the universal chloroplast expression vector pLD CtV2 (5.8 kb) to form the final Escherichia coli and tobacco shuttle vector pLD-BD Cry2Aa2 operon (9.8 kb) (Fig. 1A). This vector should be able to transform chloroplast genomes of several plant species because the flanking sequences are highly conserved among higher plants 11,12 . This vector contains the 16S ribosomal RNA (rRNA) promoter (Prrn) driving the aadA gene (aminoglycoside 3′-adenylyltransferase) for spectinomycin selection and the three genes of the cry2Aa2 operon. The terminator is the psbA 3′ region from the tobacco chloroplast genome from a gene coding for the photosystem II reaction center component. The 16S rRNA promoter is one of the strong chloroplast promoters recognized by...

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“…This poses a serious drawback when engineering multiple genes [28]. For example, to express the polyhydroxybutyrate polymer or Guy's 13 antibody, single genes were first introduced into individual plants, which were then backcrossed to reconstitute the entire pathway or the complete protein [29,30]. Similarly, in a seven-year effort, three genes were introduced in rice to generate a biosynthetic pathway for β-carotene expression [31].…”

Section: Engineering Bacterial Operons Via Chloroplast Genomesmentioning

confidence: 99%

Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology

Daniell

Khan

Allison

2002

Trends in Plant Science

324

153

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Chloroplast genomes defied the laws of Mendelian inheritance at the dawn of plant genetics, and continue to defy the mainstream approach to biotechnology, leading the field in an environmentally friendly direction. Recent success in engineering the chloroplast genome for resistance to herbicides, insects, disease and drought, and for production of biopharmaceuticals, has opened the door to a new era in biotechnology. The successful engineering of tomato chromoplasts for high-level transgene expression in fruits, coupled to hyper-expression of vaccine antigens, and the use of plant-derived antibiotic-free selectable markers, augur well for oral delivery of edible vaccines and biopharmaceuticals that are currently beyond the reach of those who need them most.Chloroplast transformation is an environmentally friendly approach to plant genetic engineering that minimizes out-crossing of transgenes to related weeds or crops [1,2] and reduces the potential toxicity of transgenic pollen to non-target insects [3]. Because the plastid genome is highly polyploid, transformation of chloroplasts permits the introduction of thousands of copies of foreign genes per plant cell, and generates extraordinarily high levels of foreign protein [3]. Chloroplast transformation vectors use two targeting sequences that flank the foreign genes and insert them, through homologous recombination, at a precise, predetermined location in the organelle genome (Fig. 1). This results in uniform transgene expression among transgenic lines and eliminates the 'position effect' often observed in nuclear transgenic plants. Gene silencing, frequently observed in nuclear transgenic plants, has not been observed in genetically engineered chloroplasts. The ability to express foreign proteins at high levels in chloroplasts and chromoplasts, and to engineer foreign genes without the use of antibiotic resistant genes [4,5],make this compartment ideal for the development of edible vaccines [6]. Moreover, the ability of chloroplasts to form disulfide bonds and to fold human proteins has opened the door to high-level production of biopharmaceuticals in plants [7]. Furthermore, foreign proteins observed to be toxic in the cytosol are non-toxic when accumulated within transgenic chloroplasts [6,8]. Chloroplast and nuclear genetic engineering are compared in Table 1.

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Generation and Assembly of Secretory Antibodies in Plants

Cited by 540 publications

References 24 publications

Manufacturing antibodies in the plant cell

Manufacturing antibodies in the plant cell

Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals

Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology

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