Transient expression in tobacco leaves of an aglycosylated recombinant antibody against the epidermal growth factor receptor

Transient expression in tobacco leaves of an aglycosylated recombinant antibody against the... When generating stably transformed transgenic plants, transient gene expression experiments are especially useful to rapidly confirm that the foreign molecule of interest is correctly assembled and retains its biological activity. TheraCIM® (CIMAB S.A., Havana) is a recombinant humanized antibody against the Epidermal Growth Factor receptor (EGF‐R), now in clinical trials for cancer therapy in Cuba and other countries. An aglycosylated version (Asn 297 was mutated for Gln 297) of this antibody was transiently expressed in tobacco leaves after vacuum‐mediated infiltration of recombinant Agrobacterium tumefaciens that carried a binary plasmid bearing the antibody heavy and light chain genes and plant regulation signals. Protein extracts from “agroinfiltrated” leaves were tested by ELISA and Western blot, showing that the fully assembled antibody was accumulated in plant tissues. The absence of plant specific glycans did not interfere in the assembling or in the activity of the plantibody, as demonstrated in this work. Indirect immunofluorescence demonstrated that the aglycosylated antibody expressed in plants recognizes the EGF‐R expressed on the surface of A431 human tumor culture cells. © 2004 Wiley Periodicals, Inc. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biotechnology and Bioengineering Wiley

Transient expression in tobacco leaves of an aglycosylated recombinant antibody against the epidermal growth factor receptor

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Publisher
Wiley
Copyright
Copyright © 2005 Wiley Periodicals, Inc., A Wiley Company
ISSN
0006-3592
eISSN
1097-0290
D.O.I.
10.1002/bit.20333
Publisher site
See Article on Publisher Site

Abstract

When generating stably transformed transgenic plants, transient gene expression experiments are especially useful to rapidly confirm that the foreign molecule of interest is correctly assembled and retains its biological activity. TheraCIM® (CIMAB S.A., Havana) is a recombinant humanized antibody against the Epidermal Growth Factor receptor (EGF‐R), now in clinical trials for cancer therapy in Cuba and other countries. An aglycosylated version (Asn 297 was mutated for Gln 297) of this antibody was transiently expressed in tobacco leaves after vacuum‐mediated infiltration of recombinant Agrobacterium tumefaciens that carried a binary plasmid bearing the antibody heavy and light chain genes and plant regulation signals. Protein extracts from “agroinfiltrated” leaves were tested by ELISA and Western blot, showing that the fully assembled antibody was accumulated in plant tissues. The absence of plant specific glycans did not interfere in the assembling or in the activity of the plantibody, as demonstrated in this work. Indirect immunofluorescence demonstrated that the aglycosylated antibody expressed in plants recognizes the EGF‐R expressed on the surface of A431 human tumor culture cells. © 2004 Wiley Periodicals, Inc.

Journal

Biotechnology and BioengineeringWiley

Published: Jan 20, 2005

Keywords: anti‐EGF receptor; aglycosylation; immunofluorescence; Nicotiana tabacum ; transient expression; vacuum infiltration

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