High level accumulation of single-chain variable fragments in the cytosol of transgenic Petunia hybrida.

The accumulation of five murine single-chain variable fragments, binding to dihydroflavonol 4-reductase, was analyzed in transgenic Petunia hybrida plants. The five scFv-encoding sequences were cloned in an optimized plant transformation vector for expression in the cytosol under control of the 35S promoter. In a transient expression assay we found that the scFv expression levels were reproducible and correlated with those in stably transformed petunia. Our results show that accumulation in the cytosol strongly depends on the intrinsic properties of the scFv fragment. Three of the five scFv fragments accumulated to unexpectedly high levels in the cytosol of the primary transformants, but no phenotypic effect could be detected. Experimental results indicate that one of the scFv fragments accumulated in the cytosol to 1% of the total soluble protein as a functional antigen-binding protein in the absence of disulphide bonds. This observation supports the idea that certain antibody fragments do not need disulphide bonds to be stable and functional. Such scFv scaffolds provide new opportunities to design scFv fragments for immunomodulation in the cytosol.

[1]  H. Gilbert.  Molecular and cellular aspects of thiol-disulfide exchange. , 2006, Advances in enzymology and related areas of molecular biology.

[2]  W. Stiekema,et al.  Improving scFv antibody expression levels in the plant cytosol 1 , 1997, FEBS letters.

[3]  U. Conrad,et al.  Seed‐specific immunomodulation of abscisic acid activity induces a developmental switch , 1997, The EMBO journal.

[4]  M. Van Montagu,et al.  Use of phage display for isolation and characterization of single‐chain variable fragments against dihydroflavonol 4‐reductase from Petunia hybrida , 1997, FEBS letters.

[5]  A. Plückthun,et al.  A natural antibody missing a cysteine in VH: consequences for thermodynamic stability and folding. , 1997, Journal of molecular biology.

[6]  M. Montagu,et al.  An Agrobacterium-mediated transient gene expression system for intact leaves , 1997 .

[7]  E. Pohl,et al.  Contribution of the intramolecular disulfide bridge to the folding stability of REIv, the variable domain of a human immunoglobulin kappa light chain. , 1996, Folding & design.

[8]  M. Van Montagu,et al.  Bacterial and plant‐produced scFv proteins have similar antigen‐binding properties , 1996, FEBS letters.

[9]  E. Weiler,et al.  Expression of a single-chain Fv antibody against abscisic acid creates a wilty phenotype in transgenic tobacco. , 1995, The Plant journal : for cell and molecular biology.

[10]  U. Conrad,et al.  High-Level Production and Long-Term Storage of Engineered Antibodies in Transgenic Tobacco Seeds , 1995, Bio/Technology.

[11]  A. Cattaneo,et al.  Redox State of Single Chain Fv Fragments Targeted to the Endoplasmic Reticulum, Cytosol and Mitochondria , 1995, Bio/Technology.

[12]  M. Reitz,et al.  Intracellular expression of antibody fragments directed against HIV reverse transcriptase prevents HIV infection in vitro , 1995, Nature Medicine.

[13]  J. Denecke,et al.  The tobacco homolog of mammalian calreticulin is present in protein complexes in vivo. , 1995, The Plant cell.

[14]  J. Bagley,et al.  Inhibition of HIV‐1 Tat‐mediated LTR transactivation and HIV‐1 infection by anti‐Tat single chain intrabodies. , 1995, The EMBO journal.

[15]  R. Sitia,et al.  The differential effects of dithiothreitol and 2-mercaptoethanol on the secretion of partially and completely assembled immunoglobulins suggest that thiol-mediated retention does not take place in or beyond the Golgi. , 1994, Molecular biology of the cell.

[16]  H. Huits,et al.  Genetic control of dihydroflavonol 4-reductase gene expression in Petunia hybrida. , 1994, The Plant journal : for cell and molecular biology.

[17]  A. Lawson,et al.  Multimerization behaviour of single chain Fv variants for the tumour-binding antibody B72.3. , 1994, Protein engineering.

[18]  M. Whitlow,et al.  Multivalent Fvs: characterization of single-chain Fv oligomers and preparation of a bispecific Fv. , 1994, Protein engineering.

[19]  A. Cattaneo,et al.  Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack , 1993, Nature.

[20]  T Prospero,et al.  "Diabodies": small bivalent and bispecific antibody fragments. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[21]  A. Plückthun.  Mono‐ and Bivalent Antibody Fragments Produced in Escherichia coli: Engineering, Folding and Antigen Binding , 1992, Immunological reviews.

[22]  V. Walbot,et al.  Identification of the motifs within the tobacco mosaic virus 5'-leader responsible for enhancing translation. , 1992, Nucleic acids research.

[23]  G. Whitelam,et al.  Synthesis of a Functional Anti–Phytochrome Single–Chain Fv Protein in Transgenic Tobacco , 1992, Bio/Technology.

[24]  R. Rycke,et al.  Plant and mammalian sorting signals for protein retention in the endoplasmic reticulum contain a conserved epitope. , 1992, The EMBO journal.

[25]  R. Glockshuber,et al.  The disulfide bonds in antibody variable domains: effects on stability, folding in vitro, and functional expression in Escherichia coli. , 1992, Biochemistry.

[26]  D. Hilvert,et al.  In vivo catalysis of a metabolically essential reaction by an antibody. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[27]  P. Benfey,et al.  The Cauliflower Mosaic Virus 35S Promoter: Combinatorial Regulation of Transcription in Plants , 1990, Science.

[28]  M. Neuberger,et al.  Expression and targeting of intracellular antibodies in mammalian cells. , 1990, The EMBO journal.

[29]  A. Hiatt,et al.  Production of antibodies in transgenic plants , 1989, Nature.

[30]  M. Van Montagu,et al.  Determination of the Processing Sites of an Arabidopsis 2S Albumin and Characterization of the Complete Gene Family. , 1988, Plant physiology.

[31]  J. Mol,et al.  An anti-sense chalcone synthase gene in transgenic plants inhibits flower pigmentation , 1988, Nature.

[32]  J. Carlson,et al.  A new means of inducibly inactivating a cellular protein , 1988, Molecular and cellular biology.

[33]  S. Rudikoff,et al.  Functional antibody lacking a variable-region disulfide bridge. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[34]  S. Munro,et al.  An hsp70-like protein in the ER: Identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein , 1986, Cell.

[35]  Jonathan D. G. Jones,et al.  High level expression of introduced chimaeric genes in regenerated transformed plants , 1985, The EMBO journal.

[36]  Marc Van Montagu,et al.  Efficient octopine Ti plasmid-derived vectors for Agrobacterium- mediated gene transfer to plants , 1985, Nucleic Acids Res..

[37]  G. Wagner,et al.  Metabolic pathways as enzyme complexes: evidence for the synthesis of phenylpropanoids and flavonoids on membrane associated enzyme complexes. , 1985, Archives of biochemistry and biophysics.

[38]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[39]  B. Satiat-Jeunemaitre,et al.  Maintenance of the exocytotic and endocytic apparatus involved in protein targeting in plant cells , 1996 .

[40]  M. D. Smith,et al.  Antibody production in plants. , 1996, Biotechnology advances.