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

Geert De Jaeger (UGent) , Emmanuel Buys, Dominique Eeckhout (UGent) , Chris De Wilde (UGent) , Anni Jacobs (UGent) , Jyoti Kapila (UGent) , Geert Angenon (UGent) , Marc Van Montagu (UGent) , Tom Gerats (UGent) and Anna Depicker (UGent)
(1999) EUROPEAN JOURNAL OF BIOCHEMISTRY. 259(1-2). p.426-434
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Abstract
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.
Keywords
FV-ANTIBODY, DISULFIDE BRIDGE, ENDOPLASMIC-RETICULUM, ANTIGEN-BINDING, ESCHERICHIA-COLI, ANTIBODY FRAGMENTS, scFv fragment, Petunia hybrida, immunomodulation, disulfide bond, Agrobacterium-mediated transient expression, PROTEIN, GENE-EXPRESSION, DIHYDROFLAVONOL 4-REDUCTASE, PLANTS

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Chicago
De Jaeger, Geert, Emmanuel Buys, Dominique Eeckhout, Chris De Wilde, Anni Jacobs, Jyoti Kapila, Geert Angenon, Marc Van Montagu, Tom Gerats, and Anna Depicker. 1999. “High Level Accumulation of Single-chain Variable Fragments in the Cytosol of Transgenic Petunia Hybrida.” European Journal of Biochemistry 259 (1-2): 426–434.
APA
De Jaeger, G., Buys, E., Eeckhout, D., De Wilde, C., Jacobs, A., Kapila, J., Angenon, G., et al. (1999). High level accumulation of single-chain variable fragments in the cytosol of transgenic Petunia hybrida. EUROPEAN JOURNAL OF BIOCHEMISTRY, 259(1-2), 426–434.
Vancouver
1.
De Jaeger G, Buys E, Eeckhout D, De Wilde C, Jacobs A, Kapila J, et al. High level accumulation of single-chain variable fragments in the cytosol of transgenic Petunia hybrida. EUROPEAN JOURNAL OF BIOCHEMISTRY. 1999;259(1-2):426–34.
MLA
De Jaeger, Geert, Emmanuel Buys, Dominique Eeckhout, et al. “High Level Accumulation of Single-chain Variable Fragments in the Cytosol of Transgenic Petunia Hybrida.” EUROPEAN JOURNAL OF BIOCHEMISTRY 259.1-2 (1999): 426–434. Print.
@article{112598,
  abstract     = {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.},
  author       = {De Jaeger, Geert and Buys, Emmanuel and Eeckhout, Dominique and De Wilde, Chris and Jacobs, Anni and Kapila, Jyoti and Angenon, Geert and Van Montagu, Marc and Gerats, Tom and Depicker, Anna},
  issn         = {0014-2956},
  journal      = {EUROPEAN JOURNAL OF BIOCHEMISTRY},
  language     = {eng},
  number       = {1-2},
  pages        = {426--434},
  title        = {High level accumulation of single-chain variable fragments in the cytosol of transgenic Petunia hybrida},
  url          = {http://dx.doi.org/10.1046/j.1432-1327.1999.00060.x},
  volume       = {259},
  year         = {1999},
}

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