A carcinoembryonic antigen-specific diabody produced in tobacco.
The feasibility of using tobacco for production of a recombinant antibody (T84.66/GS8 diabody) directed against the carcinoembryonic antigen (CEA) and used for tumor imaging was investigated. Two constructs were generated for targeting the protein either to the apoplast or to the endoplasmic reticulum. Expression of the diabody in tobacco leaves after vacuum-assisted infiltration of engineered Agrobacteria (agro-infiltration) and in regenerated transgenic tobacco plants was analyzed and compared. Results in terms of protein expression and accumulation between both systems showed a good correlation. His6-tagged T84.66 diabody was readily purified from agro-infiltrated tobacco leaves and from transgenic plants by immobilized metal ion affinity chromatography. The purified protein was analyzed by polyacrylamide gel electrophoresis, Western blot, gel filtration, electrospray mass spectrometry, direct and competition ELISA, electrophoretic mobility shift assay, and staining of CEA-positive colon adenocarcinoma cell line LS174T. Our results demonstrate that tobacco is a competent production system for this clinically relevant diabody.
Vaquero
Carmen
C
Institut für Biologie VII (Molekulare Biotechnologie, RWTH Aachen, 52074 Aachen, Germany. vaquero@molbiotech.rwth-aachen.de
Sack
Markus
M
Schuster
Flora
F
Finnern
Ricarda
R
Drossard
Jürgen
J
Schumann
Detlef
D
Reimann
Andreas
A
Fischer
Rainer
R
D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D000918:Antibody Specificity; D002272:Carcinoembryonic Antigen / Q000276:immunology*; D002846:Chromatography, Affinity; D003110:Colonic Neoplasms / Q000276:immunology*; D005240:Feasibility Studies; D006801:Humans; D008856:Microscopy, Fluorescence; D018515:Plant Leaves / Q000378:metabolism / Q000382:microbiology; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D012231:Rhizobium / Q000235:genetics; D014026:Nicotiana / Q000033:anatomy & histology / Q000235:genetics* / Q000382:microbiology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; D011994:Recombinant Proteins