title: Fluorescent protein fusions to a human immunodeficiency virus monoclonal antibody reveal its intracellular transport through the plant endomembrane system.
abstract: In order to further understand the production and intracellular trafficking of pharmaceutical proteins in plants, the light and heavy chains (LC and HC) of the human immunodeficiency virus neutralizing monoclonal antibody 2G12 were fused to fluorescent proteins [Venus and monomeric red fluorescent protein (mRFP)] to enable the visualization of their passage through the plant cell. Co-expression of LC and HC with various markers of the endomembrane system demonstrated that LC fusions were found in mobile punctate structures, which are likely to be pre-vacuolar compartments (PVCs) as a proportion of the LC fusions were found to be located in the vacuole. In addition, apoplast labelling was also observed with a 2G12LC-RFP fusion. The HC fusion expressed alone was found only in the endoplasmic reticulum (ER). When the LC and HC fusions were expressed together, they were found to co-locate to larger punctate structures, which were morphologically distinct from any observed on expression of LC or HC alone. These structures appeared to be in close association with the ER and their labelling partially overlapped with PVC marker fluorescence, but no increase in apoplast labelling was observed. Co-immunoprecipitation data demonstrated that the presence of the fluorescent proteins did not affect the assembly of the antibody, and also showed the association of BiP with the antibody chains. The antigen-binding activity of the Venus-fused 2G12 antibody was confirmed by enzyme-linked immunosorbent assay.
authors.affiliation: School of Life Sciences, Oxford Brookes University, Gipsy Lane, Headington, Oxford, UK.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Irons
authors.forename: James
authors.initials: J
authors.lastname: Nuttall
authors.forename: Doreen M
authors.initials: DM
authors.lastname: Floss
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Frigerio
authors.forename: Amanda M
authors.initials: AM
authors.lastname: Kotzer
authors.forename: Chris
authors.initials: C
authors.lastname: Hawes
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D001666:Binding Sites, Antibody; D000080908:Broadly Neutralizing Antibodies; D004797:Enzyme-Linked Immunosorbent Assay; D015483:HIV Antibodies / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000032:analysis; D004364:Pharmaceutical Preparations / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000378:metabolism; D012231:Rhizobium; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D014170:Transformation, Genetic; D000097573:Red Fluorescent Protein
chemical_list: D000911:Antibodies, Monoclonal; D000080908:Broadly Neutralizing Antibodies; D015483:HIV Antibodies; D008164:Luminescent Proteins; D004364:Pharmaceutical Preparations; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C529157:2G12 monoclonal antibody