title: Enhanced recovery of a secreted mammalian protein from suspension culture of genetically modified tobacco cells.
abstract: Increasing the level of recovery of mammalian proteins secreted by a genetically modified Nicotiana tabacum was explored in suspension culture. As a model protein system, a mouse monoclonal antibody heavy chain gamma (MAb HC) with an antigen specificity for p-azophenylarsonate was used. Consistent with findings for other plant cell suspension culture systems expressing proteins with mammalian leader sequences, the synthesized mouse MAb HC was secreted through the plasma membrane. In addition, the majority of the MAb HC was also secreted through the cell wall into the growth medium. However, efficient recovery of the protein was only possible when the protein stabilizing agent, polyvinylpyrrolidone (PVP) was present in the plant cell growth medium. The presence of PVP increased the recovered concentration of secreted protein 35-fold from 0.010 to 0.36 micrograms protein/ml culture medium. Biological activity of the approximately 50-kDa MAb HC polypeptide was demonstrated by arsonate affinity matrix binding as determined by Western blot analysis. In addition to antigen binding activity, the secreted protein also exhibited reactivity to protein G, a protein which specifically binds mouse IgG. These findings are important because they demonstrate that culture conditions can significantly influence the concentration of a biologically active foreign protein secreted from plant cells into the media of suspension cultures. The ability to increase the efficiency of mammalian protein production in plant suspension culture systems should provide significant advantage over protein production in intact transgenic plants which require cultivation, harvesting, and expensive extraction procedures to obtain nonsecreted foreign proteins.
authors.affiliation: Department of Microbiology, Washington State University, Pullman 99164-4233, USA.
authors.forename: N S
authors.initials: NS
authors.lastname: Magnuson
authors.forename: P M
authors.initials: PM
authors.lastname: Linzmaier
authors.forename: J W
authors.initials: JW
authors.lastname: Gao
authors.forename: R
authors.initials: R
authors.lastname: Reeves
authors.forename: G
authors.initials: G
authors.lastname: An
authors.forename: J M
authors.initials: JM
authors.lastname: Lee
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D015152:Blotting, Northern; D015153:Blotting, Western; D002458:Cell Fractionation; D002478:Cells, Cultured; D002999:Clone Cells / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000276:immunology; D051379:Mice; D030821:Plants, Genetically Modified / Q000235:genetics; D010947:Plants, Toxic; D011205:Povidone / Q000494:pharmacology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism*; D014170:Transformation, Genetic / Q000235:genetics; D010132:p-Azobenzenearsonate / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D011994:Recombinant Proteins; D010132:p-Azobenzenearsonate; D011205:Povidone