Abstract
Agroinfiltration of plant leaves with binary vectors carrying a gene of interest within a plant viral vector is a rapid and efficient method for protein production in plants. Previously, we constructed a self-replicating vector, pA7248AMV, based on the genetic elements of potato virus X (PVX), and have shown that this vector can be used for the expression of recombinant proteins in Nicotiana benthamiana. However, this vector is almost 18 kb long and therefore not convenient for genetic manipulation. Furthermore, for efficient expression of the target protein it should be co-agroinfiltrated with an additional binary vector expressing a suppressor of post-transcriptional gene silencing. Here, we improved this expression system by creating the novel pEff vector. Its backbone is about 5 kb shorter than the original vector and it contains an expression cassette for the silencing suppressor, P24, from grapevine leafroll-associated virus-2 alongside PVX genetic elements, thus eliminating the need of co-agroinfiltration. The pEff vector provides green fluorescent protein expression levels of up to 30% of total soluble protein. The novel vector was used for expression of the influenza vaccine candidate, M2eHBc, consisting of an extracellular domain of influenza virus M2 protein (M2e) fused to hepatitis B core antigen. Using the pEff system, M2eHBc was expressed to 5–10% of total soluble protein, several times higher than with original pA7248AMV vector. Plant-produced M2eHBc formed virus-like particles in vivo, as required for its use as a vaccine. The new self-replicating pEff vector could be used for fast and efficient production of various recombinant proteins in plants.
Highlights
Recombinant proteins, including those for medical purposes, can be produced in different expression systems, including bacteria, yeast, plants, and mammalian cells
To create an efficient plant expression vector we combined the advantages of different systems, namely, a binary vector reduced in backbone size, a self-replicating potato virus X (PVX)-based vector with translational enhancer, and various suppressors of Post-transcriptional gene silencing (PTGS)
Upon agroinfiltration of plant tissue and transfer of transfer a defined segment of DNA (T-DNA) into plant cells, the mRNA transcription from the 35S promoter results in synthesis of the viral vector RNA, the viral vector is replicated in the infected cells, the subgenomic RNA encoding the desired gene is synthesized at high level, and the target protein is expressed
Summary
Recombinant proteins, including those for medical purposes, can be produced in different expression systems, including bacteria, yeast, plants, and mammalian cells. Recombinant proteins can be produced in plants by genetic transformation or transient expression (Pogue et al, 2002; Gleba et al, 2007; Lico et al, 2008). The level of expression of target proteins by transgenic plants is usually low, leading to a high cost of products due to the difficulty of their purification (Edelbaum et al, 1992; Kusnadi et al, 1997). Transient gene expression provides a rapid alternative to the generation of stably transformed plants (Kapila et al, 1997). The advantages of such plant biofactories are the ease of manipulation, speed, low cost, and usually higher protein yield per weight of plant tissue
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