title: High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector.
abstract: We constructed a novel autonomously replicating gene expression shuttle vector, with the aim of developing a system for transiently expressing proteins at levels useful for commercial production of vaccines and other proteins in plants. The vector, pRIC, is based on the mild strain of the geminivirus Bean yellow dwarf virus (BeYDV-m) and is replicationally released into plant cells from a recombinant Agrobacterium tumefaciens Ti plasmid. pRIC differs from most other geminivirus-based vectors in that the BeYDV replication-associated elements were included in cis rather than from a co-transfected plasmid, while the BeYDV capsid protein (CP) and movement protein (MP) genes were replaced by an antigen encoding transgene expression cassette derived from the non-replicating A. tumefaciens vector, pTRAc. We tested vector efficacy in Nicotiana benthamiana by comparing transient cytoplasmic expression between pRIC and pTRAc constructs encoding either enhanced green fluorescent protein (EGFP) or the subunit vaccine antigens, human papillomavirus subtype 16 (HPV-16) major CP L1 and human immunodeficiency virus subtype C p24 antigen. The pRIC constructs were amplified in planta by up to two orders of magnitude by replication, while 50% more HPV-16 L1 and three- to seven-fold more EGFP and HIV-1 p24 were expressed from pRIC than from pTRAc. Vector replication was shown to be correlated with increased protein expression. We anticipate that this new high-yielding plant expression vector will contribute towards the development of a viable plant production platform for vaccine candidates and other pharmaceuticals.
authors.affiliation: Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Observatory, South Africa.
authors.forename: Guy L
authors.initials: GL
authors.lastname: Regnard
authors.forename: Richard P
authors.initials: RP
authors.lastname: Halley-Stott
authors.forename: Fiona L
authors.initials: FL
authors.lastname: Tanzer
authors.forename: Inga I
authors.initials: II
authors.lastname: Hitzeroth
authors.forename: Edward P
authors.initials: EP
authors.lastname: Rybicki
mesh_terms: D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism; D017791:Geminiviridae / Q000235:genetics*; D018628:Gene Dosage; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016655:HIV Core Protein p24 / Q000235:genetics* / Q000378:metabolism; D009856:Oncogene Proteins, Viral / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D012231:Rhizobium / Q000235:genetics; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes
chemical_list: D036022:Capsid Proteins; D016655:HIV Core Protein p24; D009856:Oncogene Proteins, Viral; C485184:enhanced green fluorescent protein; C514799:p24 protein, Human Immunodeficiency Virus Type 1; D049452:Green Fluorescent Proteins; C063317:L1 protein, Human papillomavirus type 16