C. Vaquero et al., Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves, P NAS US, 96(20), 1999, pp. 11128-11133
Citations number
46
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
To evaluate the expression of different forms of a tumor-specific antibody
in plants, we adapted a recently described Agrobacterium-mediated transient
expression system. A recombinant single-chain Fv antibody (scFvT84.66) and
a full-size mouse/human chimeric antibody (cT84.66) derived from the paren
tal murine mAb T84.66 specific for the human carcinoembryonic antigen were
engineered into a plant expression vector. Chimeric T84.66 heavy and light
chain genes were constructed by exchanging the mouse light and heavy chain
constant domain sequences with their human counterparts and cloned into two
independent plant expression vectors. In vivo assembly of full-size cT84.6
6 was achieved by simultaneous expression of the light and heavy chains aft
er vacuum infiltration of tobacco leaves with two populations of recombinan
t Agrobacterium. Upscaling the transient system permitted purification of f
unctional recombinant antibodies from tobacco leaf extracts within a week.
His6-tagged scFvT84.66 was purified by immobilized metal affinity chromatog
raphy and cT84.66 by protein A affinity chromatography, Sufficient amounts
of recombinant antibodies were recovered for detailed characterization by S
DS/PAGE, Western blotting, and ELISA.