Synthesis of a Functional Anti–Phytochrome Single–Chain Fv Protein in Transgenic Tobacco

Owen, M. R. L.; Gandecha, Atul R.; Cockburn, Bill; Whitelam, Garry C.

doi:10.1038/nbt0792-790

Cited by 168 publications

(74 citation statements)

References 21 publications

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“…Other studies on scFv expression in rect ELISA. Only in two GV-AS and two GV-SSAS young plants also report low cytoplasmic accumulation [7,12,13]. leaf extracts a significant signal was obtained; it was calcuThis can be partly explained by the fact that in the reducing lated that the antigen-binding signal of the accumulated scFv environment of the cytosol the stabilising intramolecular diin the GV-AS transformants and in the GV-SSAS transforsulphide bridges are not formed efficiently.…”

Section: Discussionmentioning

confidence: 99%

“…The monoclonal IgG1 antibody MAK33 [14] is directed against the way, plant cell metabolism can be investigated [7,8] and/or human creatine kinase-MM (CK-MM). A MAK33 scFv-encoding sequence was constructed by polymerase chain reaction (PCR) acspecifically modified to obtain or omit certain products.…”

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

“…1A). The resulting sion of functional scFv fragments in the cytoplasm [7,9], apophagemid was named pBAS2. plast [11], and ER [8,12] mating.…”

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

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Bacterial and plant‐produced scFv proteins have similar antigen‐binding properties

et al. 1996

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A gene encoding a single-chain variable (scFv)signal peptide sequence was compared, lower or even no acantibody fragment was expressed as a cytoplasmic and cumulation of the cytosol-targeted protein was observed, both endoplasmic reficulum-targeted protein in transgenic tobacco in leaves [11,12] and in seeds [13]. This suggests a lower staplants. In both cases, the scFv accumulated up to 0.01% of total bility of scFv proteins in the cytoplasm. Yet, intracellular soluble protein (TSP). The same scFv fragment was also expression of an anti-artichoke mottled crinkle virus scFv in produced in the periplasm of Escherichia coll. Measurement of Nicotiana benthamiana generated plants with scFv accumulathe affinity by ELISA indicates that the affinity of the tion levels up to 0.1% of TSP, which resulted in reduced inbacterially made scFv is about 80-fold lower than that of the fection incidence and delayed symptom development [9]. parental Fab fragment. The results suggest that the affinity of the However, few data are available on the efficiency of antigenplant-produced scFv fragments is reduced to a similar extent, binding scFv fragment synthesis and on the affinity of the implying that all the plant-produced scFv fragments are antigen produced scFv fragments for the cognate antigen. binding.To investigate the properties of plant-produced scFv mole- The accumulation levels were determined and the antigenbinding properties of the plant-produced scFv fragments were compared to those of a bacterially produced scFv.

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Section: Discussionmentioning

confidence: 99%

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

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Bacterial and plant‐produced scFv proteins have similar antigen‐binding properties

et al. 1996

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“…A complimentary approach to analyzing phytochrome function has recently been developed by Whitelam ancl coworkers and involves the transgenic expression of antiphytochrome single-chain Fv proteins (Owen et al, 1992). These proteins contain only the variable domains (VH and VI.)…”

Section: Expression Of Phytochrome In Transcenic Plantsmentioning

confidence: 99%

Illuminating Phytochrome Functions (There Is Light at the End of the Tunnel)

Vierstra

1993

Plant Physiol.

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Because green plants depend on light as a primary source of energy, they have evolved an array of mechanisms to measure light intensity, direction, duration, and spectral quality. Plants use such information, in a process commonly referred to as photomorphogenesis, to capture light more effectively and to entrain their life cycle to the climatic seasons (Kendrick and Kronenberg, 1986). Many diverse responses are under light control; these include seed germination, tropic responses, chloroplast development, stem growth, pigmentation, stomatal opening, flowering, and senescence. An interesting concept, not often considered by many, is that plants have the ability to detect light quality, which imparts a crude form of color vision that is used by plants preferring full sun to avoid shading by other plants.Light perception is initiated by at least three different photoreceptors used either singly or in tandem: phytochrome, which exists in two photochromic forms, Pr and Pfr, absorbing red and far-red light, respectively; cryptochrome, a blue/ UV-A-absorbing pigment; and a UV-B-absorbing pigment. Of these three, phytochrome is the predominant and bestcharacterized receptor (Furuya, 1989;Quail, 1991). However, despite more than 40 years of intense research since its discovery, we still do not know how phytochrome functions at the molecular level. Thankfully, this situation is rapidly changing. Recent applications of powerful molecular and genetic techniques have finally begun to illuminate the mechanism of phytochrome action and to dissect its signal transduction chains. The purpose of this review is to highlight recent discoveries concerning phytochrome structure and function to show that there is now light at the end of the tunnel. Various laboratories are also finding that a multitude of phytochromes exist, each with potentially different functions, which makes the phytochrome transduction system even more complex than previously realized.

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“…When fed to animals, the compound causes hyperestrogenism with symptoms such as enlargement of the uterus and nipples, vulvar swelling, vaginal prolapse, and infertility (16,23). In the last 10 years, the expression of specific antibodies or antibody fragments in plants has attracted great interest (6,15,21,30,36,37), and it has shown some potential in improving plant resistance against pathogens (33) and in altering plant metabolic pathways (1,27). Recently, we developed a singlechain Fv (scFv) antibody with high affinity for zearalenone (38).…”

mentioning

confidence: 99%

Expression of a Functional Antizearalenone Single-Chain Fv Antibody in Transgenic Arabidopsis Plants

Yuan

Pestka

et al. 2000

Appl Environ Microbiol

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The efficacy of cloning a recombinant mycotoxin antibody in plants was tested using Arabidopsis as a model. An antizearalenone single-chain Fv (scFv) DNA fragment was first cloned in the newly constructed phage display vector (pEY.5) and then recloned in the plant transformation vector pKYLX71::35S 2 . After transformation, constructs of antizearalenone scFv were introduced into immature Arabidopsis seeds via Agrobacterium tumefaciens mediation by vacuum infiltration. Only plants transformed with the construct containing a PR-1b signal peptide sequence produced transgenic offspring. The antizearalenone scFv "plantibody" from these transgenic plants bound zearalenone with a high affinity (50% inhibitory concentration, 11.2 ng/ml) that was comparable to that of bacterially produced scFv antibody and the parent monoclonal antibody (MAb). By electron microscopic immunogold labeling, the presence of antizearalenone scFv was detected mainly in the cytoplasm and only occasionally outside the cell. Like bacterially produced scFv antibody, antizearalenone scFv plantibody exhibited greater sensitivity to methanol destabilization than did the parent MAb. The sensitivity of antizearalenone scFv plantibody to acidic disassociation was similar to the sensitivities of bacterially produced scFv antibody and MAb. Expression of specific plantibodies in crops might be useful for neutralizing mycotoxins in animal feeds and for reducing mycotoxin-associated plant diseases.Zearalenone [6-(10-hydroxy-6-oxo-trans-1-undecenyl)-␤-resorcylic acid lactone] is a mycotoxin produced by members of the genus Fusarium after infection of corn and small grains (14,24,25). When fed to animals, the compound causes hyperestrogenism with symptoms such as enlargement of the uterus and nipples, vulvar swelling, vaginal prolapse, and infertility (16,23). In the last 10 years, the expression of specific antibodies or antibody fragments in plants has attracted great interest (6,15,21,30,36,37), and it has shown some potential in improving plant resistance against pathogens (33) and in altering plant metabolic pathways (1,27). Recently, we developed a singlechain Fv (scFv) antibody with high affinity for zearalenone (38). To explore the possibility of using "plantibodies" to neutralize mycotoxin through passive immunization of animals in their feed, as a first step we used the newly cloned antizearalenone scFv DNA fragment to transform Arabidopsis plants. In this report, we demonstrate that expression of the antizearalenone scFv gene in transgenic Arabidopsis plants leads to the accumulation of a soluble scFv plantibody with high affinity for the mycotoxin zearalenone. MATERIALS AND METHODSGeneral. All chemicals and solvents were reagent grade or better. Chemicals were purchased from Sigma Chemical Company (St. Louis, Mo.) unless otherwise noted. All DNA manipulations, if not described, were carried out by standard procedures (28).Construction of scFv cloning vector. scFv, a single-chain fragment of the antibody variable region antigen-binding protein, is c...

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Synthesis of a Functional Anti–Phytochrome Single–Chain Fv Protein in Transgenic Tobacco

Cited by 168 publications

References 21 publications

Bacterial and plant‐produced scFv proteins have similar antigen‐binding properties

Bacterial and plant‐produced scFv proteins have similar antigen‐binding properties

Illuminating Phytochrome Functions (There Is Light at the End of the Tunnel)

Expression of a Functional Antizearalenone Single-Chain Fv Antibody in Transgenic Arabidopsis Plants

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