Affinity-purification of a TMV-specific recombinant full-size antibody from a transgenic tobacco suspension culture

A TMV-specific full-size murine IgG-2b/kappa antibody (mAb24) was expressed in a Nicotiana tabacum cv. Petite Havana SR1 suspension culture (P9s), whi ch was derived from a stably transformed transgenic plant (P9). The integra tion of an N-terminal murine leader peptide directed the assembled immunogl obulin for secretion. However, in suspension culture, the full-size recombi nant antibody, rAb24, was retained by the plant cell wall and was not prese nt 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 ac id supplementation of the culture medium. For purification of the recombina nt 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 wit h cross-flow filtration (step 1) followed by protein A affinity chromatogra phy (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 pla nt 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 pu rified antibody was indistinguishable from its murine counterpart, indicati ng the potential of plant cell suspension cultures as bio-reactors for the production of recombinant antibodies. (C) 1999 Elsevier Science B.V. All ri ghts reserved.