title: Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants.
abstract: This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformedNicotiana tabacum Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy's 13 antibody heavy and light chain mutant combinations were expressed transiently inN. tabacumand demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.-Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants.
authors.affiliation: Molecular Immunology Unit, The Institute for Infection and Immunity, St. George's, University of London, London, United Kingdom.
authors.forename: Verena K
authors.initials: VK
authors.lastname: Hehle
authors.affiliation: Molecular Immunology Unit, The Institute for Infection and Immunity, St. George's, University of London, London, United Kingdom.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Paul
authors.affiliation: Molecular Immunology Unit, The Institute for Infection and Immunity, St. George's, University of London, London, United Kingdom.
authors.forename: Victoria A
authors.initials: VA
authors.lastname: Roberts
authors.affiliation: Molecular Immunology Unit, The Institute for Infection and Immunity, St. George's, University of London, London, United Kingdom.
authors.forename: Craig J
authors.initials: CJ
authors.lastname: van Dolleweerd
authors.affiliation: Molecular Immunology Unit, The Institute for Infection and Immunity, St. George's, University of London, London, United Kingdom jma@sgul.ac.uk.
authors.forename: Julian K-C
authors.initials: JK
authors.lastname: Ma
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D010447:Peptide Hydrolases
keywords: antibody engineering; degradation; proteolysis