title: Highly efficient targeting and accumulation of a F(ab) fragment within the secretory pathway and apoplast of Arabidopsis thaliana.
abstract: To further improve antibody production in plants, constructs were designed to minimize transgene silencing and to retain a F(ab) fragment within the secretory pathway of transgenic Arabidopsis thaliana plants. The levels of antibody accumulation suggest that placing the sequences that encode Fd and light chain under the control of nonidentical 3' regions reduces susceptibility to post-transcriptional gene silencing compared with when the individual polypeptide-encoding sequences are placed under the control of identical 3' regions. High levels of accumulation (up to 6% of total soluble protein) were found for both secreted and intracellularly targeted antibody fragments. Immunofluorescence microscopic analysis showed that F(ab) fragments devoid of any additional C-terminal sequence were efficiently secreted, whereas retention of F(ab) fragments within the endomembrane system of the secretory pathway was achieved by C-terminal fusion of the DIKDEL sequence to the antibody light chain. Furthermore, analysis by immunoprecipitation and ELISA showed that intracellular retention of antibody fragments did not affect antigen-binding activity, and more than 80% of the isolated antibody fragments were found to bind antigen. Taken together, our results provide improvements to the technology of recombinant antibody production in transgenic plants.
authors.affiliation: Vakgroep Moleculaire Genetica, Departement Plantengenetica, Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Universiteit Gent, K.L. Ledeganckstraat, Gent, Belgium.
authors.forename: K
authors.initials: K
authors.lastname: Peeters
authors.forename: C
authors.initials: C
authors.lastname: De Wilde
authors.forename: A
authors.initials: A
authors.lastname: Depicker
mesh_terms: D000941:Antigens / Q000378:metabolism; D017360:Arabidopsis / Q000737:chemistry*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D020868:Gene Silencing; D005822:Genetic Vectors; D015151:Immunoblotting; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D007128:Immunoglobulin Fragments; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D030821:Plants, Genetically Modified; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012323:RNA Processing, Post-Transcriptional; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D000941:Antigens; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins