ENHANCED RECOVERY OF A SECRETED MAMMALIAN PROTEIN FROM SUSPENSION-CULTURE OF GENETICALLY-MODIFIED TOBACCO CELLS

Increasing the level of recovery of mammalian proteins secreted by a g enetically modified Nicotiana tabacum was explored in suspension cultu re. As a model protein system, a mouse monoclonal antibody heavy chain gamma (MAb HC) with an antigen specificity for p-azophenylarsonate wa s used. Consistent with findings for other plant cell suspension cultu re systems expressing proteins with mammalian leader sequences, the sy nthesized mouse MAb HC was secreted through the plasma membrane. In ad dition, the majority of the MAb HC was also secreted through the cell wall into the growth medium. However, efficient recovery of the protei n was only possible when the protein stabilizing agent, polyvinylpyrro lidone (PVP) was present in the plant cell growth medium. The presence of PVP increased the recovered concentration of secreted protein 35-f old from 0.010 to 0.36 mu g protein/ml culture medium. Biological acti vity of the similar to 50-kDa MAb HC polypeptide was demonstrated by a rsonate affinity matrix binding as detemined by Western blot analysis, In addition to antigen binding activity, the secreted protein also ex hibited reactivity to protein G, a protein which specifically binds mo use IgG;. These findings are important because they demonstrate that c ulture conditions can significantly influence the concentration of a b iologically active foreign protein secreted from plant cells into the media of suspension cultures. The ability to increase the efficiency o f mammalian protein production in plant suspension culture systems sho uld provide significant advantage over protein production in intact tr ansgenic plants which require cultivation, harvesting, and expensive e xtraction procedures to obtain nonsecreted foreign proteins, (C) 1996 Academic Press, Inc.