Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves.
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 parental 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 cT84.66 was achieved by simultaneous expression of the light and heavy chains after vacuum infiltration of tobacco leaves with two populations of recombinant Agrobacterium. Upscaling the transient system permitted purification of functional recombinant antibodies from tobacco leaf extracts within a week. His6-tagged scFvT84.66 was purified by immobilized metal affinity chromatography and cT84.66 by protein A affinity chromatography. Sufficient amounts of recombinant antibodies were recovered for detailed characterization by SDS/PAGE, Western blotting, and ELISA.
Vaquero
C
C
Institut für Biologie I (Botanik/Molekulargenetik), Rheinisch-Westfälische Technische Hochschule Aachen, Worringerweg 1, D-52074 Aachen, Germany.
Sack
M
M
Chandler
J
J
Drossard
J
J
Schuster
F
F
Monecke
M
M
Schillberg
S
S
Fischer
R
R
D000818:Animals; D000912:Antibodies, Neoplasm / Q000096:biosynthesis* / Q000302:isolation & purification; D015153:Blotting, Western; D002272:Carcinoembryonic Antigen / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000302:isolation & purification; D051379:Mice; D010947:Plants, Toxic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D014026:Nicotiana / Q000235:genetics*
D000912:Antibodies, Neoplasm; D002272:Carcinoembryonic Antigen; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C055966:immunoglobulin Fv