title: Development of a single-replicon miniBYV vector for co-expression of heterologous proteins.
abstract: In planta production of recombinant proteins, including vaccine antigens and monoclonal antibodies, continues gaining acceptance. With the broadening range of target proteins, the need for vectors with higher performance is increasing. Here, we have developed a single-replicon vector based on beet yellows virus (BYV) that enables co-delivery of two target genes into the same host cell, resulting in transient expression of each target. This BYV vector maintained genetic stability during systemic spread throughout the host plant, Nicotiana benthamiana. Furthermore, we have engineered a miniBYV vector carrying the sequences encoding heavy and light chains of a monoclonal antibody (mAb) against protective antigen (PA) of Bacillius anthracis, and achieved the expression of the full-length functional anti-PA mAb at ~300 mg/kg of fresh leaf tissue. To demonstrate co-expression and functionality of two independent proteins, we cloned the sequences of the Pfs48/45 protein of Plasmodium falciparum and endoglycosidase F (PNGase F) from Flavobacterium meningosepticum into the miniBYV vector under the control of two subgenomic RNA promoters. Agroinfiltration of N. benthamiana with this miniBYV vector resulted in accumulation of biologically active Pfs48/45 that was devoid of N-linked glycosylation and had correct conformation and epitope display. Overall, our findings demonstrate that the new BYV-based vector is capable of co-expressing two functionally active recombinant proteins within the same host cell.
authors.affiliation: Fraunhofer USA Center for Molecular Biotechnology, 9 Innovation Way, Suite 200, Newark, DE, 19711, USA.
authors.forename: Alex
authors.initials: A
authors.lastname: Prokhnevsky
authors.forename: Tarlan
authors.initials: T
authors.lastname: Mamedov
authors.forename: Brett
authors.initials: B
authors.lastname: Leffet
authors.forename: Rahila
authors.initials: R
authors.lastname: Rahimova
authors.forename: Ananya
authors.initials: A
authors.lastname: Ghosh
authors.forename: Vadim
authors.initials: V
authors.lastname: Mett
authors.forename: Vidadi
authors.initials: V
authors.lastname: Yusibov
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology; D001408:Bacillus anthracis / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity; D001428:Bacterial Vaccines / Q000235:genetics / Q000276:immunology; D045247:Chryseobacterium; D017790:Closterovirus / Q000235:genetics*; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D005822:Genetic Vectors; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012093:Replicon; D014026:Nicotiana / Q000235:genetics
chemical_list: D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001428:Bacterial Vaccines; D000939:Epitopes; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C084847:pfs48-45 protein, Plasmodium falciparum; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase