Evaluation of immunoglobulins from plant cells.
Expression of cDNA constructs encoding full-length mouse immunoglobulin chains with their native leader sequences or fusion constructs substituting the native leader with a pre-pro sequence derived from Saccharomyces cerevisiae yielded blocked N-termini on the gamma chain or the correct amino terminal sequence on the mature kappa chain. Lectin binding assays revealed that assembled immunoglobulin complexes contained a glycosylated heavy chain. The attached glycan was resistant to digestion by endoglycosidase H and its lectin binding pattern was distinguishable from that of the mammalian glycan. The results indicated processing of the immunoglobulin carbohydrate in the tobacco Golgi to yield a complex oligosaccharide. Secretion of antibody by protoplasts isolated from regenerated transgenic plants or from suspension callus cells was demonstrated by pulse-chase labeling experiments. When purified, the tobacco-produced antibody was found to possess the antigen binding and catalytic properties of the murine monoclonal antibody. Kinetic parameters (Km, Ki, Vmax, and kcat) of the tobacco-derived antibody were comparable to those of the mouse-derived antibody. The results in general show that the endomembrane system of tobacco cells possesses cognate mechanisms for the recognition of diverse leader sequences. These signals can be used to initiate the assembly, processing, and secretion by plant cells of complex foreign proteins.
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
M B
MB
Hein
Y
Y
Tang
D A
DA
McLeod
K D
KD
Janda
A
A
Hiatt
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D000941:Antigens / Q000276:immunology; D002384:Catalysis; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D006031:Glycosylation; D007142:Immunoglobulin gamma-Chains / Q000378:metabolism; D007136:Immunoglobulins / Q000235:genetics* / Q000276:immunology; D000072235:Mating Factor; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics; D010944:Plants / Q000235:genetics / Q000276:immunology*; D010947:Plants, Toxic; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014026:Nicotiana / Q000276:immunology; D014170:Transformation, Genetic
D000911:Antibodies, Monoclonal; D000941:Antigens; D007142:Immunoglobulin gamma-Chains; D007136:Immunoglobulins; D010455:Peptides; D021382:Protein Sorting Signals; D000072235:Mating Factor; D004247:DNA