Secretion of a functional single-chain Fv protein in transgenic tobacco plants and cell suspension cultures

Firek, Simon; Draper, John; Owen, M. R. L.; Gandecha, Atul R.; Cockburn, Bill; Whitelam, Garry C.

doi:10.1007/bf00021540

Cited by 133 publications

(42 citation statements)

References 26 publications

(8 reference statements)

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“…Indeed, several mants was 2.5% and 5%, respectively, of the signal obtained reports document higher accumulation levels when the scFv for corresponding amounts of Fab fragment. In the mature fragments were directed towards the oxidising environment of leaf extracts from seven GV-AS and four GV-SSAS transforthe ER [11][12][13]. However, another scFv protein appeared to be mants antigen-binding scFv could be detected.…”

Section: Discussionmentioning

confidence: 99%

“…The resulting sion of functional scFv fragments in the cytoplasm [7,9], apophagemid was named pBAS2. plast [11], and ER [8,12] mating. Transformation of Nicotiana tabacum SR1 was done by leaf 14900×g for 10 min at 4°C and the supernatant was centrifuged disc infection as described by [21] and transgenic GV-AS and GVagain for 10 min for young leaves or for 10 rain and 5 min for mature SSAS plants were selected on hygromycin-containing (50 gg/ml) medleaves.…”

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

“…

Abstract 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.

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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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“…To overcome these problems other expression systems such as mammalian (Dorai et al, 1994;Jost et al, 1994), insect (Putlitz et al, 1990), yeast (Bowdish et al, 1991;Davis et al, 1991), plant (Firek et al, 1993), and in vitro translation systems (Nicholls et al, 1993) have been used to produce rAb fragments (compared in (Verma et al, 1998)). The main advantage of yeast is that it is eukaryotic, has well understood genetics, and can be manipulated and cultivated easily and inexpensively.…”

Section: Introductionmentioning

confidence: 99%

High-yield expression of the recombinant, atrazine-specific Fab fragment K411B by the methylotrophic yeast Pichia pastoris

Lange

Schmitt

Schmid

2001

Journal of Immunological Methods

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In this report, we describe the high-yield secretory expression (~ 40 mg l -1 ) of pure, atrazinespecific Fab fragments (K411B) from P. pastoris that was achieved by co-integration of the genes encoding the heavy and light chains ( Crude culture supernatant was used to study the binding properties of the Fab fragment toward different s-triazines by means of competitive ELISA: the IC 50 value for the detection of atrazine was determined from the standard curve as 3 µg l -1 , which is one magnitude higher than the value obtained with the parental mAb K4E7 but equals that obtained when the same Fab fragment was expressed in E. coli cells. In addition, the cross-reactivity pattern of the Fab from Pichia is comparable to that of E. coli and the parental mAb K4E7.

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“…rAbs are efficiently folded and assembled within the endoplasmic reticulum (ER) of plant cells (4)(5)(6) and retain the antigen binding properties of the antibodies produced by plasma or hybridoma cells (2,5,(7)(8)(9). Since the first report of antibody expression in transgenic plants (7), different engineered antibodies have been produced successfully, including full-size antibodies (8)(9)(10)(11), Fab fragments (12), scFvs (13)(14)(15)(16)(17)(18)(19)(20)(21), and single-domain antibodies (22).…”

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confidence: 99%

Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves

Vaquero

Sack

Chandler

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

219

139

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To evaluate the expression of different forms of a tumor-specific antibody in plants, we adapted a recently described Agrobacterium-mediated transient expression system. A recombinant single-chain Fv antibody (scFvT84.66) and a full-size mouse͞human chimeric antibody (cT84.66) derived from the parental murine mAb T84.66 specific for the human carcinoembryonic antigen were engineered into a plant expression vector. Chimeric T84.66 heavy and light chain genes were constructed by exchanging the mouse light and heavy chain constant domain sequences with their human counterparts and cloned into two independent plant expression vectors. In vivo assembly of full-size cT84.66 was achieved by simultaneous expression of the light and heavy chains after vacuum infiltration of tobacco leaves with two populations of recombinant Agrobacterium. Upscaling the transient system permitted purification of functional recombinant antibodies from tobacco leaf extracts within a week. His6-tagged scFvT84.66 was purified by immobilized metal affinity chromatography and cT84.66 by protein A affinity chromatography. Sufficient amounts of recombinant antibodies were recovered for detailed characterization by SDS͞PAGE, Western blotting, and ELISA.Monoclonal antibodies are essential tools in biology, biochemistry, and medicine. Their high affinity and specificity make them invaluable for diagnostic and therapeutic applications. However, the therapeutic use of murine mAbs is limited because they elicit a human anti-mouse antibody response and large amounts of antibody are required for therapy. These limitations may be overcome by engineering humanized antibodies and by producing these proteins in plants.The human anti-mouse antibody response can be reduced by using recombinant antibody (rAb) technology to replace the murine light and heavy chain constant domains with the corresponding human domains and the remaining murine variable domains to maintain the antigen specificity and affinity of the original mAb. A second approach is to use single-chain Fv antibody fragments (scFvs) where the constant domains have been removed and the variable domains are joined by a flexible linker (1). Compared with the full-size antibodies, scFvs display better tumor penetration and faster serum clearance but exhibit no effector functions. A critical step in testing the potential therapeutic use of these molecules is the development of a reproducible and efficient method for large-scale antibody production.Plants are potentially the most economical system for largescale production of rAbs (2, 3). rAbs are efficiently folded and assembled within the endoplasmic reticulum (ER) of plant cells (4-6) and retain the antigen binding properties of the antibodies produced by plasma or hybridoma cells (2,5,(7)(8)(9). Since the first report of antibody expression in transgenic plants (7), different engineered antibodies have been produced successfully, including full-size antibodies (8-11), Fab fragments (12), scFvs (13-21), and single-domain antibodies (22).Regene...

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Secretion of a functional single-chain Fv protein in transgenic tobacco plants and cell suspension cultures

Cited by 133 publications

References 26 publications

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

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

High-yield expression of the recombinant, atrazine-specific Fab fragment K411B by the methylotrophic yeast Pichia pastoris

Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves

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