Efficient Transient Expression of Recombinant Proteins in Plants by the Novel pEff Vector Based on the Genome of Potato Virus X

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.

Plant-Produced Recombinant Influenza A Virus Candidate Vaccine Based on Flagellin Linked to Conservative Fragments of M2 Protein and Hemagglutintin

“…Similar expression levels of M2e-containing proteins in plants have been achieved in other studies [24][25][26]. These values were lower than those obtained in the expression of flagellin with four attached copies of the M2e of the avian influenza virus (0.5-1 mg/g before purification [23]), but they were sufficient for the production of recombinant protein quantities necessary for immunogenicity testing and estimation of protective action in animal studies. Protein samples were analyzed using SDS-PAGE and Western blotting with M2e-specific antibodies.…”

“…The pEff viral vector [23] was used to express the target proteins in N. benthamiana plants. This expression vector is based on the genome of PVX and includes the 5'-untranslated region of the PVX genome, the RNA-dependent RNA polymerase gene, the first promoter of the subgenomic RNA, the 5'-untranslated region of the alfalfa mosaic virus (AMV) RNA 4, acting as a translational enhancer, the green fluorescent protein gene (gfp), the last 60 nucleotides of the coat protein gene of PVX, and the 3'-untranslated region of the PVX genome.…”

Transient protein expression in tobacco BY-2 plant cell packs using single and multi-cassette replicating vectors

“…Combining key elements of the different expression systems, which are not mutually exclusive, could contribute to development of more effective systems. Recently, the advantages of pEAQ vectors, PVX-based vectors, and P24 silencing suppressor were successfully combined and resulted in a new vector, pEff, for transient expression of recombinant proteins in plants (Mardanova et al 2017). Similarly, the combination of a P19 cassette with the CPMVhypertranslatable (CPMV-HT) UTRs and a geminivirus-based LIR-SIR-LIR system represents another possible improvement (Peyret and Lomonossoff 2015).…”

Rapid High-Yield Transient Expression of Swine Hepatitis E ORF2 Capsid Proteins in Nicotiana benthamiana Plants and Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope

“…In the current study, we describe the efficient transient expression of HEV ORF2 genotype 3 capsid proteins in N. benthamiana using two expression vectors: the Cowpea Mosaic Virus (CPMV)-based vector pEAQ-HT [24,25] and the potato X virus (PVX) -based vector pEff [26]. pEAQ-HT is a nonreplicating system which uses the production of a highly-translatable mRNA to achieve high level expression.…”

“…This construction is inserted between the 35S promoter and 35S terminator. Additionally, the vector contains an expression cassette for the silencing suppressor, P24, from grapevine leafroll-associated virus-2 [26].…”