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High-level expression of a single-chain Fv fragment (scFv) antibody in transgenic pea seeds
J. Plant Physiol. 158. 529 – 533 (2001) Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp
High-level expression of a single-chain Fv fragment (scFv) antibody in transgenic pea seeds* Isolde Saalbach, Martin Giersberg, Udo Conrad** IPK Gatersleben, Corrensstraße 3, D-06466 Gatersleben Received January 8, 2001 · Accepted January 12, 2001
Summary The field pea (Pisum sativum L.) is a protein-rich crop and pea seeds are well suited for the production of recombinant proteins. Here we show, that recombinant antibodies can be accumulated in transgenic pea seeds in a homozygous transgenic line up to 2 % of total soluble seed protein. The expression was controlled by the seed-specific USP promoter and the transgenic single-chain Fv antibody protein was retended in the endoplasmic reticulum. The stable inheritance, shown by investigation of the high-level accumulation in the R3 offspring is another important feature of this new antibody production system. The suitability of transgenic pea seeds as an economically potent production system for recombinant antibodies is clearly demonstrated by our results. Key words: field pea – recombinant antibodies – expression system – seed-specific promoter Abbreviations: ABA abscisic acid. – ER endoplasmic reticulum. – GUS β-glucuronidase. – LeB leguminB from Vicia faba. – TSP total soluble protein. – USP unknown seed protein from Vicia faba
Introduction In the last years, recombinant antibody technology has been developed and improved to provide effective tools to produce specific and high-affine antibodies for therapy in human and veterinary medicine as well as for sensitive analysis in medicine, biochemistry and pest control in plant breeding (Russel 1999, Kerschbaumer and Himmler 1999, Ziegler 1999). Several types of recombinant antibodies have been developed (Pluckthun and Pack 1997). Single-chain Fv antibodies, consisting of VH and VK chains connected by a flex* This paper is more focussed than the oral presentation at the meeting since a more general review has been published recently (Artsaenko et al. 1999). ** E-mail corresponding author: [email protected]
ible linker are the common type of this new therapeutical and analytical tools (Bird et al.1988). Representative libraries and efficient screening methods have been developed to isolate specific scFv against diverse antigens (De Wildt et al. 2000). ScFv’s are of specific interest for use in medicine, because they lack constant domains and therefore they are less immunogenic than complete immunoglobulins. Better penetration of tissues and more rapid distribution in the body are further benefits of these small potentially therapeutic proteins. Recombinant antibodies could be produced in microbial expression systems as E. coli and yeast (Fischer et al. 1999) at lower costs than by use of hybridoma cell culture facilities, but the lack of correct folding leads to serious problems, when production technologies have to be developed. Transgenic plants as production system of recombinant proteins provide several benefits such as high expression levels and organ-specific ex0176-1617/01/158/04-529 $ 15.00/0
530
Isolde Saalbach, Martin Giersberg, Udo Conrad
pression controlled by specific promoters, several cell compartments proven for stable accumulation of transgenic proteins, easy methods to scale up production systems just by seeding on bigger fields and an already existing agricultural seeding, harvesting and storage technology. Recombinant antibodies, especially scFv have been produced in several compartments of plant cells to high levels (for review see Conrad and Fiedler 1998, Fischer et al. 2000). ScFv have been produced in seeds and in potato tubers to store them for extended periods (Fiedler and Conrad 1995, Artsaenko et al. 1998). Highest accumulation has been achieved by retention of scFv in the endoplasmic reticulum (Artsaenko et al. 1995). Seeds are of specifc interest, because they contain high amounts of protein and could be easily handled and stored. Even for seeds, the ER retention technology combined with the use of a potent seed-specific promoter provided optimal scFv accumulation levels, as shown for transgenic tobacco (Fiedler et al. 1997). After basic research in tobacco and Arabidopsis the developed technology has to be transferred to suitable crops to establish economically potent expression systems (Stöger et al. 2000). Legume grains are of specific interest because of their high protein content. Recently, expression of the scFv in pea seeds has been shown by use of a seed-specific promoter. Nine µg per gram fresh weight of a functional scFv have been achieved. The principial utility of field pea seeds for production of pharmaceutical macromolecules has been demonstrated (Perrin et al. 2000). However, these results are far below seed-specific expression levels demonstrated for tobacco. The highest expression of scFv in tobacco seeds has been achieved by use of the seedspecific USP promoter combined with ER retention (Phillips et al. 1997). We therefore tried to adapt this expression system
using the same promoter and scFv to field pea. We show here, that comparable levels of stable expression of scFv could be achieved in transgenic field pea, too. These strongly improved expression levels open the way for even economically efficient production of recombinant antibodies in field pea seeds.
Materials and Methods Bacterial strains and Ti plasmid For these studies, an anti-ABA-scFv gene, described by Artsaenko et al. (1995) was used. The basic expression cassette contained the seed-specific USP promoter, the LeB4 signal sequence, the anti-ABA scFv gene, the ER retention signal coding sequence and the c-myctag coding sequence (Phillips et al. 1997, Fig. 1). The GUS sequence was removed from the vector plasmid pGPTV-bar (Becker et al. 1992) by cleavage with EcoRI and SmaI. The religated plasmid was called pPTV-bar. The expression cassette described above was cloned into the HindIII site of the vector resulting in the plasmid pPTV-bar⬋aABA. Cloning steps were performed according to standard procedures (Sambrook et al. 1989). The resulting expession vector pPTVbar⬋aABA was transformed into the hypervirulent Agrobacterium tumefaciens strain EHA 105 by electroporation.
Plant transformation A plant transformation system for fodder pea varieties was developed based on the protocol of Schroeder et al. (1993). Here «Erbi» as a fodder pea (normal leaf) variety planted in Germany was used. Explants for transformation were cut from the embryonic axis of immature seeds. For this purpose pods were sterilized, seeds were removed from the pods and testae were excised. These seeds were precultivated for 1 day. For preparation of explants the root end of
Figure 1. A: Schematic picture of the expression cassette B: Map of the expression vector pPTV-bar⬋aABA. c-myc: c-myc-tag coding sequence for immunological detection; LeB4: signal peptide coding sequence from the legumin B4 gene; bar: BASTA-resistance coding gene.
Antibody production in transgenic peas each segment was cut off and the epicotyl and apical meristem regions were sliced transversely into 3 – 5 segments. Following inoculation, the sclices were co-cultivated for 3 – 4 days at 21 ˚C with a 16 h photoperiod. After co-cultivation explants were washed three to four times with sterile water. The callus induction medium and the shoot development were based on the protocol of Schroeder et al. (1993). When the developing shoots were over 20 mm in length, they were selected on 10 mg/L phosphinothricine for some days again. The resistent shoots were grafted. Grafting of putative transformed shoots was achieved using the rootstock of «Erbi» seedlings in vitro. After 6–10 days the plants were adapted to soil and grown up in a climatic chamber. When primary transformants developed to a stage of 4 – 6 normal nodes, herbizide leaf painting with a solution of BASTA (Hoechst) at 0.5 % was performed. Inheritance of the indroduced trangene was also monitored by herbicide application as described before. One part of seeds harvested from each of the primary transformants was grown up in a climatic chamber and leaf painting was performed as described.
Analysis of transgenic pea seeds Extract preparation, SDS electrophoresis and Western blot analysis were performed as described in Conrad et al. (1997). ScFv concentration was roughly quantitated by theTINA-programme (Raytest). For the segregation analysis of the scFv expression seeds of the first generation were imbibed and a small slice were cut and used for extract preparation. Segregation analysis according the BASTA resistance was done by herbizide leaf painting as described above. DNA was prepared from leaf tissues according the method of Dellaporta et al. (1984). Restriction analysis, DNA-transfer and hybridisation at high stringency conditions were performed by standard protocols (Sambrook et al. 1989).
Results Immature seeds of pea were transformed with the construct pPTV-bar⬋aABA and transgenic plants were produced as described above. Grafted R0 plants were tested by leaf painting and BASTA-resistant plants were selected. Seeds of 12 independent transgenic plants were investigated by Western blotting and 10 plants were found to accumulate scFv. In Figure 2, scFv expression in seeds of two R0 plants is shown.
Figure 2. ScFv expression in seeds of transgenic pea plants (R0). 1: 5 µg seed extract plant E11; 2: 10 µg seed extract plant E11; 3: 20 µg seed extract plant E11; 4: 5 µg seed extract plant E17; 5: 10 µg seed extract plant E17; 6: 20 µg seed extract plant E17; 7: 25 ng purified anti-ABA-scFv (from transgenic tobacco); 8: 50 ng purified anti-ABA-scFv; 9: 10 ng purified anti-ABA-scFv; 10: 20 µg seed extract from BU seeds, 17 DAP (Phillips et al. 1997). The scFv was detected by anti-c-myc antibody, anti-MausIG-POD and ECL.
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Generally, scFv accumulation up to 1 % of total soluble protein was detected in seeds of R0 plants. From 10 lines offspring was investigated according BASTA resistance by leaf painting and according scFv expression by Western blots from seed protein extracts. Therefore, from 7 to 22 R1 seeds one slice was used for scFv detection and the rest of the seed was used to grow a plant, that was each tested by leaf painting according BASTA resistance (Table 1). The number of inserts was estimated from the segregation pattern of BASTA resistance and antibody expression. As shown in Table 1, segregation of both properties is roughly similar. In case of line 1, DNA was prepared from leaves of R1 plants and cleaved with EcoRI. EcoRI sites are not present within the transgenic DNA fragment between left and right border (4.6 kBp). From the size of single band deteted by southern blot (6.5 kBp Fig. 3) we therefore conclude, that one copy was integrated as one insert into the pea genome. From this transgenic plant a homozygous line was selected by self-pollination. The offspring showed no segregation for BASTA resistance. In several R3 seeds of this line the scFv accumulation has been tested by Western blot. The expression level was about 2 % of TSP, as shown in Figure 4. Comparable scFv expression has been confirmed for other R3 seeds of line 1 (data not shown). Generally, among 12 BASTA-resistant lines 9 showed stable inheritance of the transgene, documented by expression analysis and BASTA resistance analysis (Table 1). In several lines the scFv antibody fragment was stably accumulated to high concentrations even in the offspring.
Discussion Seeds and green tissues of tobacco and Arabidopsis as well as potato tubers have been shown to be suitable production tools for various recombinant antibodies (for review see Conrad and Fiedler 1998). Seed-specific expression of scFv was shown for tobacco by use of the seed-specific legumin B4 promoter (Fiedler and Conrad 1995) and by use of the seedspecific USP promoter (Phillips et al. 1997, Conrad et al. 1998). Recently, active pharmaceutical scFv have been pro-
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Isolde Saalbach, Martin Giersberg, Udo Conrad
Table 1. Segregation analysis of BASTA resistance and scFv expression in the offspring of transgenic pea plants. Roplants (stable) 1 5 7 9 11 12 16 17 18 19
Basta resistance
Anti-ABA scFv antibody
No. of tested seeds (R1)
segregation of R1
No. of tested seeds (R1)
segregation of R1
22 11 10 7 10 15 8 8 13 8
19 : 3 11 : 0 10 : 0 1:6 9:1 13 : 2 6:2 6:2 10 : 3 5:3
22 4 4 3 4 5 4 5 4
19 : 3 4:0 4:0 0:3 4:0 5:0 3:1 3:2 4:0 n. d.
No. of inserts
1 ≥2 ≥2 chimaeric 2 2 1 1 1 1?
anti-ABA scFv antibody content (percent of TSP) 1–2 n.d. n.d. n.d. n.d. 0.2 – 0.3 0.2 – 0.5 0.2 – 0.5 0.05 – 0.1 0.05 – 0.1
shown here, a stably producing high expression line could selected out of 12 independent transformants. Seeds of this line showed high expression until R3. The segregation data of the scFv expression and the BASTA resistance (both 19 : 3 in R1,) show, that a single insertion ocurred in the genome of this line. This was confirmed by Southern data (Fig. 3). Here we show, that even out of a few independent transgenic pea lines, high-producer lines for scFv could be isolated. This opens the way for the efficient production of different lines producing several therapeutical scFv for given projects in parallel. The high expression level is important for minimizing production costs in case of direct application and for the development of efficient down stream processing procedures. Furthermore, cultivation, harvesting and storage technology for pea seeds are well established. In further experiments down stream processing technologies have to be developed
Figure 3. Detection of the integrated expression cassette in transgenic peas by Southern analysis. The pea genomic DNA was cleaved with EcoRI and hybridised to the HindIII fragment coding for the scFv (Fig. 1). Different sublines of E1 were tested. uc: uncleaved DNA.
duced in seeds of cereal crops, where up to 1.4 µg/g dry weight have been obtained (Stöger et al. 2000). In a rather new paper Perrin et al. (2000) reported about the seed-specific expression of an active scFv in pea seeds under control of a seed-specific legumin A promoter. Pea seeds could express the scFv up to 9 µg/g. Phillips et al. (1997) did not only find rather high expression of the scFv in several transgenic tobacco lines, but also detected high-level of expression in the seeds of more than one quarter of the independent lines, when expression was controlled by the USP promoter. Therefore, we wanted to test, if this legume promoter could be used for the establishment of an efficient production system for several recombinant antibodies in pea seeds. As we have
Figure 4. ScFv expression in transgenic seeds of the pea line E1 (R3). 3, 4, 5: extracts from different seeds of R3 plants. Standard: purified anti-ABA-scFv. The scFv was detected by anti-c-myc antibody, antiMausIG-POD and ECL.
Antibody production in transgenic peas for pea seeds. The system we have established will be applied to therapeutical recombinant antibodies, too.
References Artsaenko O, Kettig B, Fiedler U, Conrad U, Düring K (1998) Potato tubers as a biofactory for recombinant antibodies. Molecular Breeding 4: 313 – 319 Artsaenko O, Peisker M, Zur Nieden U, Fiedler U, Weiler EW, Müntz K, Conrad U (1995) Expression of single-chain Fv antibody against abscisic acid creates a wilty phenotype in transgenic tobacco. Plant J 8: 745–750 Artsaenko O, Phillips J, Fiedler U, Peisker M, Conrad U (1999) Intracellular immunomodulation in plants – a new tool for the investigation of phytohormones. In: Harper K, Ziegler A (eds) Recombinant Antibodies–Applications in Plant Science and Plant Pathology. Taylor & Francis, London, pp 145–156 Becker D, Kemper E, Schell J, Masterson R (1992) New plant binary vectors with selectable markers located proximal to the left T-DNA border. PMB 20: 1195–1197 Bird DE, Hardmann KD, Jacobsohn JW, Johnson S, Kaufman BM, Lee SM (1988) Single-chain antigen-binding proteins. Science 242: 242 – 426 Conrad U, Fiedler U (1998) Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity. PMB 38: 101–109 Conrad U, Fiedler U, Artsaenko O, Phillips J (1997) Single-chain Fv Antibodies Expressed in Plants. In: Cunningham C, Porter A (eds) Recombinant Proteins from Plants: Production and Isolation of Clinically Useful Compounds. Humana Press, Totowa, pp 89–102 Conrad U, Fiedler U, Artsaenko O, Phillips J (1998) High level and stable accumulation of single-chain Fv antibodies in plant storage organs. J Plant Physiol 152: 708–711 Dellaporta SL, Wood J, Hicks JB (1984) Maize DNA miniprep. In: Malmberg R, Messing J, Sussex I (eds) Molecular Biology of Plants: A Laboratory Course Manual. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, pp 36 – 37 De Wildt RM, Mundy CR, Gorick BD, Tomlinson IM (2000) Antibody arrays for high-throughput screening of antibody – antigen interaction. Nat Biotechnol 18: 989 – 994 Fiedler U, Phillips J, Artsaenko O, Conrad U (1997) Optimization of scFv production in transgenic plants. Immunotechnology 3: 205 – 216
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Fiedler U, Conrad U (1995) High-level production and long-term storage of engineered antibodies in transgenic tobacco seeds. Bio/ technology 13: 1090–1093 Fischer R, Drossard J, Emans N, Commandeur U, Hellwig S (1999) Towards molecular farming in the future: pichia pastoris-based production of single-chain antibody fragments. Biotechnol Appl Biochem 30: 17– 20 Fischer R, Hoffmann K, Schillberg S, Emans N (2000) Antibody production by molecular farming in plants. J Biol Regul Homeost Agents 14: 83 – 92 Kerschbaumer R, Himmler G (1999) Dedicated expression vectors for the production of diagnostic reagents. In: Harper K, Ziegler A (eds) Recombinant Antibodies–Applications in Plant Science and Pathology. Taylor & Francis, London, pp 57–75 Perrin Y, Vaquero C, Gerrard I, Sack M, Drossard J, Stöger E, Christou P, Fischer R (2000) Transgenic pea seeds as bioreactors for the production of a single-chain Fv fragment (scFv) antibody used in cancer diagnostic and therapy. PMB 6: 345 – 352 Phillips J, Artsaenko O, Fiedler U, Horstmann C, Mock H-P, Müntz K, Conrad U (1997) Seed-specific immunomodulation of abscisic acid activity induces a developmental switch. EMBO J 16: 4489 – 4611 Pluckthun A, Pack P (1997) New protein engineering approaches to multivalent and bispecific antibody fragments. Immunotechnology 3 (2): 83–105 Russel DA (1999) Feasibility of antibody production in plants for human therapeutic use. In: Hammond J, McGarvey P, Yusibov V (eds) Plant Biotechnology – New Products and Applications. Springer-Verlag, New York, pp 44 – 58 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory Press, Cold Spring Harbour Schroeder HE, Schotz T, Wardley-Richardson D, Spencer D, Higgins TJV (1993) Transformation and regeneration of two cultivars of pea (Pisum sativum). Plant Physiol 101: 751–757 Stöger E, Vaquero C, Torres E, Sack M, Nicholson L, Drossard J, Williams S, Keen D, Perrin Y, Christou P, Fischer R (2000) Cereal crops as viable production and storage system for pharmaceutical scFv antibodies. PMB 42: 583 – 590 Ziegler A (1999) Cloning of pre-existing Hybridomas-Production of Novel Diagnostic Tools for Plant Pathology. In: Harper K, Ziegler A (eds) Recombinant Antibodies–Applications in Plant Science and Pathology. Taylor & Francis, London, pp 23 – 32
High-level expression of a single-chain Fv fragment (scFv) antibody in transgenic pea seeds* Isolde Saalbach, Martin Giersberg, Udo Conrad** IPK Gatersleben, Corrensstraße 3, D-06466 Gatersleben Received January 8, 2001 · Accepted January 12, 2001
Summary The field pea (Pisum sativum L.) is a protein-rich crop and pea seeds are well suited for the production of recombinant proteins. Here we show, that recombinant antibodies can be accumulated in transgenic pea seeds in a homozygous transgenic line up to 2 % of total soluble seed protein. The expression was controlled by the seed-specific USP promoter and the transgenic single-chain Fv antibody protein was retended in the endoplasmic reticulum. The stable inheritance, shown by investigation of the high-level accumulation in the R3 offspring is another important feature of this new antibody production system. The suitability of transgenic pea seeds as an economically potent production system for recombinant antibodies is clearly demonstrated by our results. Key words: field pea – recombinant antibodies – expression system – seed-specific promoter Abbreviations: ABA abscisic acid. – ER endoplasmic reticulum. – GUS β-glucuronidase. – LeB leguminB from Vicia faba. – TSP total soluble protein. – USP unknown seed protein from Vicia faba
Introduction In the last years, recombinant antibody technology has been developed and improved to provide effective tools to produce specific and high-affine antibodies for therapy in human and veterinary medicine as well as for sensitive analysis in medicine, biochemistry and pest control in plant breeding (Russel 1999, Kerschbaumer and Himmler 1999, Ziegler 1999). Several types of recombinant antibodies have been developed (Pluckthun and Pack 1997). Single-chain Fv antibodies, consisting of VH and VK chains connected by a flex* This paper is more focussed than the oral presentation at the meeting since a more general review has been published recently (Artsaenko et al. 1999). ** E-mail corresponding author: [email protected]
ible linker are the common type of this new therapeutical and analytical tools (Bird et al.1988). Representative libraries and efficient screening methods have been developed to isolate specific scFv against diverse antigens (De Wildt et al. 2000). ScFv’s are of specific interest for use in medicine, because they lack constant domains and therefore they are less immunogenic than complete immunoglobulins. Better penetration of tissues and more rapid distribution in the body are further benefits of these small potentially therapeutic proteins. Recombinant antibodies could be produced in microbial expression systems as E. coli and yeast (Fischer et al. 1999) at lower costs than by use of hybridoma cell culture facilities, but the lack of correct folding leads to serious problems, when production technologies have to be developed. Transgenic plants as production system of recombinant proteins provide several benefits such as high expression levels and organ-specific ex0176-1617/01/158/04-529 $ 15.00/0
530
Isolde Saalbach, Martin Giersberg, Udo Conrad
pression controlled by specific promoters, several cell compartments proven for stable accumulation of transgenic proteins, easy methods to scale up production systems just by seeding on bigger fields and an already existing agricultural seeding, harvesting and storage technology. Recombinant antibodies, especially scFv have been produced in several compartments of plant cells to high levels (for review see Conrad and Fiedler 1998, Fischer et al. 2000). ScFv have been produced in seeds and in potato tubers to store them for extended periods (Fiedler and Conrad 1995, Artsaenko et al. 1998). Highest accumulation has been achieved by retention of scFv in the endoplasmic reticulum (Artsaenko et al. 1995). Seeds are of specifc interest, because they contain high amounts of protein and could be easily handled and stored. Even for seeds, the ER retention technology combined with the use of a potent seed-specific promoter provided optimal scFv accumulation levels, as shown for transgenic tobacco (Fiedler et al. 1997). After basic research in tobacco and Arabidopsis the developed technology has to be transferred to suitable crops to establish economically potent expression systems (Stöger et al. 2000). Legume grains are of specific interest because of their high protein content. Recently, expression of the scFv in pea seeds has been shown by use of a seed-specific promoter. Nine µg per gram fresh weight of a functional scFv have been achieved. The principial utility of field pea seeds for production of pharmaceutical macromolecules has been demonstrated (Perrin et al. 2000). However, these results are far below seed-specific expression levels demonstrated for tobacco. The highest expression of scFv in tobacco seeds has been achieved by use of the seedspecific USP promoter combined with ER retention (Phillips et al. 1997). We therefore tried to adapt this expression system
using the same promoter and scFv to field pea. We show here, that comparable levels of stable expression of scFv could be achieved in transgenic field pea, too. These strongly improved expression levels open the way for even economically efficient production of recombinant antibodies in field pea seeds.
Materials and Methods Bacterial strains and Ti plasmid For these studies, an anti-ABA-scFv gene, described by Artsaenko et al. (1995) was used. The basic expression cassette contained the seed-specific USP promoter, the LeB4 signal sequence, the anti-ABA scFv gene, the ER retention signal coding sequence and the c-myctag coding sequence (Phillips et al. 1997, Fig. 1). The GUS sequence was removed from the vector plasmid pGPTV-bar (Becker et al. 1992) by cleavage with EcoRI and SmaI. The religated plasmid was called pPTV-bar. The expression cassette described above was cloned into the HindIII site of the vector resulting in the plasmid pPTV-bar⬋aABA. Cloning steps were performed according to standard procedures (Sambrook et al. 1989). The resulting expession vector pPTVbar⬋aABA was transformed into the hypervirulent Agrobacterium tumefaciens strain EHA 105 by electroporation.
Plant transformation A plant transformation system for fodder pea varieties was developed based on the protocol of Schroeder et al. (1993). Here «Erbi» as a fodder pea (normal leaf) variety planted in Germany was used. Explants for transformation were cut from the embryonic axis of immature seeds. For this purpose pods were sterilized, seeds were removed from the pods and testae were excised. These seeds were precultivated for 1 day. For preparation of explants the root end of
Figure 1. A: Schematic picture of the expression cassette B: Map of the expression vector pPTV-bar⬋aABA. c-myc: c-myc-tag coding sequence for immunological detection; LeB4: signal peptide coding sequence from the legumin B4 gene; bar: BASTA-resistance coding gene.
Antibody production in transgenic peas each segment was cut off and the epicotyl and apical meristem regions were sliced transversely into 3 – 5 segments. Following inoculation, the sclices were co-cultivated for 3 – 4 days at 21 ˚C with a 16 h photoperiod. After co-cultivation explants were washed three to four times with sterile water. The callus induction medium and the shoot development were based on the protocol of Schroeder et al. (1993). When the developing shoots were over 20 mm in length, they were selected on 10 mg/L phosphinothricine for some days again. The resistent shoots were grafted. Grafting of putative transformed shoots was achieved using the rootstock of «Erbi» seedlings in vitro. After 6–10 days the plants were adapted to soil and grown up in a climatic chamber. When primary transformants developed to a stage of 4 – 6 normal nodes, herbizide leaf painting with a solution of BASTA (Hoechst) at 0.5 % was performed. Inheritance of the indroduced trangene was also monitored by herbicide application as described before. One part of seeds harvested from each of the primary transformants was grown up in a climatic chamber and leaf painting was performed as described.
Analysis of transgenic pea seeds Extract preparation, SDS electrophoresis and Western blot analysis were performed as described in Conrad et al. (1997). ScFv concentration was roughly quantitated by theTINA-programme (Raytest). For the segregation analysis of the scFv expression seeds of the first generation were imbibed and a small slice were cut and used for extract preparation. Segregation analysis according the BASTA resistance was done by herbizide leaf painting as described above. DNA was prepared from leaf tissues according the method of Dellaporta et al. (1984). Restriction analysis, DNA-transfer and hybridisation at high stringency conditions were performed by standard protocols (Sambrook et al. 1989).
Results Immature seeds of pea were transformed with the construct pPTV-bar⬋aABA and transgenic plants were produced as described above. Grafted R0 plants were tested by leaf painting and BASTA-resistant plants were selected. Seeds of 12 independent transgenic plants were investigated by Western blotting and 10 plants were found to accumulate scFv. In Figure 2, scFv expression in seeds of two R0 plants is shown.
Figure 2. ScFv expression in seeds of transgenic pea plants (R0). 1: 5 µg seed extract plant E11; 2: 10 µg seed extract plant E11; 3: 20 µg seed extract plant E11; 4: 5 µg seed extract plant E17; 5: 10 µg seed extract plant E17; 6: 20 µg seed extract plant E17; 7: 25 ng purified anti-ABA-scFv (from transgenic tobacco); 8: 50 ng purified anti-ABA-scFv; 9: 10 ng purified anti-ABA-scFv; 10: 20 µg seed extract from BU seeds, 17 DAP (Phillips et al. 1997). The scFv was detected by anti-c-myc antibody, anti-MausIG-POD and ECL.
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Generally, scFv accumulation up to 1 % of total soluble protein was detected in seeds of R0 plants. From 10 lines offspring was investigated according BASTA resistance by leaf painting and according scFv expression by Western blots from seed protein extracts. Therefore, from 7 to 22 R1 seeds one slice was used for scFv detection and the rest of the seed was used to grow a plant, that was each tested by leaf painting according BASTA resistance (Table 1). The number of inserts was estimated from the segregation pattern of BASTA resistance and antibody expression. As shown in Table 1, segregation of both properties is roughly similar. In case of line 1, DNA was prepared from leaves of R1 plants and cleaved with EcoRI. EcoRI sites are not present within the transgenic DNA fragment between left and right border (4.6 kBp). From the size of single band deteted by southern blot (6.5 kBp Fig. 3) we therefore conclude, that one copy was integrated as one insert into the pea genome. From this transgenic plant a homozygous line was selected by self-pollination. The offspring showed no segregation for BASTA resistance. In several R3 seeds of this line the scFv accumulation has been tested by Western blot. The expression level was about 2 % of TSP, as shown in Figure 4. Comparable scFv expression has been confirmed for other R3 seeds of line 1 (data not shown). Generally, among 12 BASTA-resistant lines 9 showed stable inheritance of the transgene, documented by expression analysis and BASTA resistance analysis (Table 1). In several lines the scFv antibody fragment was stably accumulated to high concentrations even in the offspring.
Discussion Seeds and green tissues of tobacco and Arabidopsis as well as potato tubers have been shown to be suitable production tools for various recombinant antibodies (for review see Conrad and Fiedler 1998). Seed-specific expression of scFv was shown for tobacco by use of the seed-specific legumin B4 promoter (Fiedler and Conrad 1995) and by use of the seedspecific USP promoter (Phillips et al. 1997, Conrad et al. 1998). Recently, active pharmaceutical scFv have been pro-
532
Isolde Saalbach, Martin Giersberg, Udo Conrad
Table 1. Segregation analysis of BASTA resistance and scFv expression in the offspring of transgenic pea plants. Roplants (stable) 1 5 7 9 11 12 16 17 18 19
Basta resistance
Anti-ABA scFv antibody
No. of tested seeds (R1)
segregation of R1
No. of tested seeds (R1)
segregation of R1
22 11 10 7 10 15 8 8 13 8
19 : 3 11 : 0 10 : 0 1:6 9:1 13 : 2 6:2 6:2 10 : 3 5:3
22 4 4 3 4 5 4 5 4
19 : 3 4:0 4:0 0:3 4:0 5:0 3:1 3:2 4:0 n. d.
No. of inserts
1 ≥2 ≥2 chimaeric 2 2 1 1 1 1?
anti-ABA scFv antibody content (percent of TSP) 1–2 n.d. n.d. n.d. n.d. 0.2 – 0.3 0.2 – 0.5 0.2 – 0.5 0.05 – 0.1 0.05 – 0.1
shown here, a stably producing high expression line could selected out of 12 independent transformants. Seeds of this line showed high expression until R3. The segregation data of the scFv expression and the BASTA resistance (both 19 : 3 in R1,) show, that a single insertion ocurred in the genome of this line. This was confirmed by Southern data (Fig. 3). Here we show, that even out of a few independent transgenic pea lines, high-producer lines for scFv could be isolated. This opens the way for the efficient production of different lines producing several therapeutical scFv for given projects in parallel. The high expression level is important for minimizing production costs in case of direct application and for the development of efficient down stream processing procedures. Furthermore, cultivation, harvesting and storage technology for pea seeds are well established. In further experiments down stream processing technologies have to be developed
Figure 3. Detection of the integrated expression cassette in transgenic peas by Southern analysis. The pea genomic DNA was cleaved with EcoRI and hybridised to the HindIII fragment coding for the scFv (Fig. 1). Different sublines of E1 were tested. uc: uncleaved DNA.
duced in seeds of cereal crops, where up to 1.4 µg/g dry weight have been obtained (Stöger et al. 2000). In a rather new paper Perrin et al. (2000) reported about the seed-specific expression of an active scFv in pea seeds under control of a seed-specific legumin A promoter. Pea seeds could express the scFv up to 9 µg/g. Phillips et al. (1997) did not only find rather high expression of the scFv in several transgenic tobacco lines, but also detected high-level of expression in the seeds of more than one quarter of the independent lines, when expression was controlled by the USP promoter. Therefore, we wanted to test, if this legume promoter could be used for the establishment of an efficient production system for several recombinant antibodies in pea seeds. As we have
Figure 4. ScFv expression in transgenic seeds of the pea line E1 (R3). 3, 4, 5: extracts from different seeds of R3 plants. Standard: purified anti-ABA-scFv. The scFv was detected by anti-c-myc antibody, antiMausIG-POD and ECL.
Antibody production in transgenic peas for pea seeds. The system we have established will be applied to therapeutical recombinant antibodies, too.
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