Affinity-purification of a TMV-specific recombinant full-size antibody from a transgenic tobacco suspension culture.
A TMV-specific full-size murine IgG-2b/K antibody (mAb24) was expressed in a Nicotiana tabacum cv. Petite Havana SR1 suspension culture (P9s), which was derived from a stably transformed transgenic plant (P9). The integration of an N-terminal murine leader peptide directed the assembled immunoglobulin for secretion. However, in suspension culture, the full-size recombinant antibody, rAb24, was retained by the plant cell wall and was not present in the culture medium. rAb24 expression reached a basal level of 15 microg per gram wet cell weight, corresponding to 0.3% of the total soluble plant cell protein. The level of rAb24 could be increased three-fold by amino acid supplementation of the culture medium. For purification of the recombinant antibody from batch-cultured tobacco suspension cells, the primary plant cell wall was partially digested by enzymatic treatment. This resulted in a total release of recombinant full-size rAb24 into the extraction buffer. A three-step procedure was used to purify the immunoglobulins, starting with cross-flow filtration (step 1) followed by protein A affinity chromatography (step 2) and gel filtration as a final purification step (step 3). This procedure gave a recovery of more than 80% of the expressed rAb24 from plant cell extracts. SDS-PAGE, IEF and immunoblot analyses demonstrated a high degree of homogeneity for the affinity-purified rAb24. An ELISA procedure demonstrated that the specificity and affinity of the protein A affinity purified antibody was indistinguishable from its murine counterpart, indicating the potential of plant cell suspension cultures as bio-reactors for the production of recombinant antibodies.
Institut für Biologie I (Botanik)/Molekulargenetik), RWTH Aachen, Germany. fischer@bio1.rwth-aachen.de
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Fischer
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Liao
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Drossard
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology / Q000302:isolation & purification*; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods*; D003470:Culture Media; D051379:Mice; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D014026:Nicotiana / Q000821:virology*; D014027:Tobacco Mosaic Virus / Q000276:immunology*
D000914:Antibodies, Viral; D003470:Culture Media; D011994:Recombinant Proteins