High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector
Article first published online: 19 NOV 2009
© 2009 The Authors. Journal compilation © 2009 Blackwell Publishing Ltd
Plant Biotechnology Journal
Volume 8, Issue 1, pages 38–46, January 2010
How to Cite
Regnard, G. L., Halley-Stott, R. P., Tanzer, F. L., Hitzeroth, I. I. and Rybicki, E. P. (2010), High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector. Plant Biotechnology Journal, 8: 38–46. doi: 10.1111/j.1467-7652.2009.00462.x
- Issue published online: 9 DEC 2009
- Article first published online: 19 NOV 2009
- Received 2 April 2009; revised 28 September 2009; accepted 5 October 2009.
- Bean yellow dwarf virus;
- replicating plant expression vector
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.