Abstract
The yield and assembly of an IgG1 antibody and its derived Fab fragment were compared inNicotiana andArabidopsis. The results obtained showed a lot of interclonal variability. For 45% of the primary transgenic calluses, antigen-binding entities represented less than 0.1% of the total soluble protein (TSP). Only two of the 103 analysed transformants contained more than 1% of antigen-binding protein, with 1.26% being the highest yield. Analogous amounts of complete antibody and Fab accumulated in primary callus tissue. Moreover, yields were in the same range for both species as far as primary callus tissue is concerned. However, the accumulation of the Fab fragment in leaf tissue of regenerated plants differed significantly betweenNicotiana andArabidopsis. The Fab fragment accumulated to only 0.044% of TSP inNicotiana leaves but up to 1.3% inArabidopsis leaves. Furthermore, both species showed differences in the assembly pattern of the complete antibody. WhereasArabidopsis contained primarily fully assembled antibodies of 150 kDa,Nicotiana showed an abundance of fragments in the 50 kDa range.
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Bauw, G., Holm Rasmussen, H., Van den Bulcke, M., Van Damme, J., Puype, M., Gesser, B., Celis, J.E. and Vandekerckhove, J. (1990) Two-dimensional gel electrophoresis, protein electroblotting and microsequencing: a direct link between proteins and genes.Electrophoresis 11, 528–36.
Benvenuto, E., Ordàs, R.J., Tavazza, R., Ancora, G., Biocca, S., Cattaneo, A. and Galeffi, P. (1991) ‘Phytoantibodies’: a general vector for the expression of immunoglobulin domains in transgenic plants.Pl. Mol. Biol. 17, 865–74.
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72, 248–54.
Breyne, P., Gheysen, G., Jacobs, A., Van Montagu, M. and Depicker, A. (1992) Effect of T-DNA configuration on transgene expression.Mol. Gen. Genet. 235, 389–96.
Buchner, J. and Rudolph, R. (1991) Renaturation, purification and characterization of recombinant Fab-fragments produced inEscherichia coli.Bio/Technology 9, 157–62.
Buckel, P., Hübner-Parajsz, C., Mattes R., Lenz, H., Haug, H. and Beaucamp, K. (1987) Cloning and nucleotide sequence of heavy- and light-chain cDNAs from a creatine-kinasespecific monoclonal antibody.Gene 51, 13–9.
Chrispeels, M.J. (1991) Sorting of proteins in the secretory system.Ann. Rev. Pl. Physiol. Pl. Mol. Biol. 42, 21–53.
De Greve, H., Dhaese, P., Seurinck, J., Lemmers, M., Van Montagu, M. and Schell, J. (1982) Nucleotide sequence and transcript map of theAgrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene.J. Mol. Appl. Genet. 1, 499–512.
De Sutter, K., Feys, V., Van de Voorde, A. and Fiers, W. (1992) Production of functionally active murine and murine::human chimeric F(ab)2 fragments in COS-1 cells.Gene 113, 223–30.
Deblaere, R., Bytebier, B., De Greve, H., Deboeck, F., Schell, J., Van Montagu, M. and Leemans, J. (1985) Efficient octopine Ti plasmid-derived vectors forAgrobacterium-mediated gene transfer to plants.Nucl. Acids Res.13, 4777–88.
Denecke, J., De Rycke, R. and Botterman, J. (1992) Plant and mammalian sorting signals for protein retention in the endoplasmic reticulum contain a conserved epitope.EMBO J.11, 2345–55.
Depicker, A., Herman, L., Jacobs, A., Schell, J. and Van Montagu, M. (1985) Frequencies of simultaneous transformation with different T-DNAs and their relevance to theAgrobacterium/plant cell interaction.Mol. Gen. Genet. 201, 477–84.
Düring, K., Hippe, S., Kreuzaler, F. and Schell, J. (1990) Synthesis and self-assembly of a functional monoclonal antibody in transgenicNicotiana tabacum.Pl. Mol. Biol. 15, 281–93.
Haas, I.G. (1991) BiP — A heat shock protein involved in immunoglobulin chain assembly.Curr. Topics Microbiol. Immunol. 167, 71–82.
Harlow, E. and Lane, D. (1988)Antibodies. A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Hein, M.B., Tang, Y., McLeod, D.A., Janda, K.D. and Hiatt, A. (1991) Evaluation of immunoglobulins from plant cells.Biotechnol. Prog. 7, 455–61.
Hiatt, A., Cafferkey, R. and Bowdish, K. (1989) Production of antibodies in transgenic plants.Nature 342, 76–8.
Horwitz, A.H., Chang, C.P., Better, M., Hellstrom, K.E. and Robinson, R.R. (1988) Secretion of functional antibody and Fab fragment from yeast cells.Proc. Natl Acad. Sci. USA 85, 8678–82.
Ingelbrecht, I.L.W., Herman, L.M.F., Dekeyser, R.A., Van Montagu, M.C. and Depicker, A.G. (1989) Different 3′ end regions strongly influence the level of gene expression in plant cells.Pl. Cell. 1, 671–80.
Krebbers, E., Herdies, L., De Clercq, A., Seurinck, J., Leemans, J., Van Damme, J., Segura, M., Gheysen, G., Van Montagu M. and Vandekerckhove, J. (1988) Determination of the processing sites of anArabidopsis 2S albumin and characterization of the complete gene family.Pl. Physiol. 87, 859–66.
Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982)Molecular cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Noiva, R. and Lennarz, W.J. (1992) Protein disulfide isomerase. A multifunctional protein resident in the lumen of the endoplasmic reticulum.J. Biol. Chem. 267, 3553–6.
Odell, J.T., Nagy, F. and Chua, N.H. (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter.Nature 313, 810–2.
Owen, M., Gandecha, A., Cockburn, B. and Whitelam, G. (1992) Synthesis of a functional anti-phytochrome singlechain Fv protein in transgenic tobacco.Bio/Technology 10, 790–4.
Peach, C. and Velten, J. (1991) Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters.Pl. Mol. Biol. 17, 49–60.
Plückthun, A. (1991) Antibody engineering: advances from the use ofEscherichia coli expression systems.Bio/Technology 9, 545–51.
Sijmons, P.C., Dekker, B.M.M., Schrammeijer, B., Verwoerd, T.C., van den Elzen, P.J.M. and Hoekema, A. (1990) Production of correctly processed human serum albumin in transgenic plants.Bio/Technology 8 217–21.
Sokal, R.R. and Rohlf, F.J. (1981).Biometry, The Principles and Practice of Statistics in Biological Research, 2nd ed. New York: W.H. Freeman and Company.
Svoboda, M., Meuris, S., Robyn, C. and Christophe, J. (1985) Rapid electrotransfer of proteins from polyacrylamide gel to nitrocellulose membrane using surface-conductive glass as anode.Anal. Biochem. 151, 16–23.
Van Lijsebettens, M., Vanderhaeghen, R. and Van Montagu, M. (1991) Insertional mutagenesis inArabidopsis thaliana: isolation of a T-DNA-linked mutation that alters leaf morphology.Theor. Appl. Genet. 81 277–84.
Weidle, U.H., Borgya, A., Mattes, R., Lenz, H. and Buckel, P. (1987) Reconstitution of functionally active antibody directed against creatine kinase from separately expressed heavy and light chains in non-lymphoid cells.Gene 51, 21–9.
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de Neve, M., De Loose, M., Jacobs, A. et al. Assembly of an antibody and its derived antibody fragment inNicotiana andArabidopsis . Transgenic Research 2, 227–237 (1993). https://doi.org/10.1007/BF01977353
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DOI: https://doi.org/10.1007/BF01977353