Affinity-purification of a TMV-specific recombinant full-size antibody from a transgenic tobacco suspension culture
| Autor: | Rainer Fischer, Jürgen Drossard, Yu-Cai Liao |
|---|---|
| Přispěvatelé: | Publica |
| Rok vydání: | 1999 |
| Předmět: |
antibody expression
transgenic plant cell suspension cultures medicine.drug_class Nicotiana tabacum Immunology transgenic plants Antibodies Viral Monoclonal antibody Chromatography Affinity law.invention Mice Affinity chromatography law Tobacco medicine Tobacco mosaic virus recombinant antibody Animals Immunology and Allergy Cells Cultured antibody purification biology Tobamovirus Plants Genetically Modified biology.organism_classification Molecular biology Recombinant Proteins Culture Media Tobacco Mosaic Virus Plants Toxic biology.protein Recombinant DNA Antibody Protein A |
| Zdroj: | Journal of Immunological Methods. 226:1-10 |
| ISSN: | 0022-1759 |
| DOI: | 10.1016/s0022-1759(99)00058-7 |
| Popis: | A TMV-specific full-size murine IgG-2b/kappa 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 mu g 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. |
| Databáze: | OpenAIRE |
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