Myriam De Neve

Assembly of an antibody and its derived antibody fragment in Nicotiana and Arabidopsis.

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.

Bacterial and plant-produced scFv proteins have similar antigen-binding properties

A gene encoding a single‐chain variable (scFv) antibody fragment was expressed as a cytoplasmic and endoplasmic reticulum‐targeted protein in transgenic tobacco plants. In both cases, the scFv accumulated up to 0.01% of total soluble protein (TSP). The same scFv fragment was also produced in the periplasm of Escherichia coli. Measurement of the affinity by ELISA indicates that the affinity of the bacterially made scFv is about 80‐fold lower than that of the parental Fab fragment. The results suggest that the affinity of the plant‐produced scFv fragments is reduced to a similar extent, implying that all the plant‐produced scFv fragments are antigen binding.

Intact antigen-binding MAK33 antibody and Fab fragment accumulate in intercellular spaces of Arabidopsis thaliana

Abstract The in planta localization of a full-size immunoglobulin G (IgG) antibody and of its derived F ab fragment were determined using immunogold electron microscopy on transgenic Arabidopsis thaliana plants. Although it is widely assumed that antibodies, following assembly in the lumen of the endoplasmic reticulum, migrate via the secretory pathway to the cell surface, until now their subcellular in planta localization has not been demonstrated. Here, we show that both IgG and F ab fragment accumulate within intercellular spaces of leaf mesophyll cells of Arabidopsis . Using immunoblot analysis on leaf intercellular fluid, we demonstrate that these microscopically detected proteins are integral and functional antibodies or F ab fragments. The fact that a full-size IgG is not physically restricted from passage through the cell wall, despite having a molecular mass of 146 kD, questions the generally accepted limitations in cell wall porosity. These results are of particular significance in any plantibody approach that intends to modulate extracellular antigen function.

Use of phage display for isolation and characterization of single-chain variable fragments against dihydroflavonol 4-reductase from Petunia hybrida

© 1997 Federation of European Biochemical Societies.