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.

Engineering, production and characterization of Spike and Nucleocapsid structural proteins of SARS–CoV-2 inNicotiana benthamianaas vaccine candidates against COVID-19

“…This system has been successfully used for rapid and cost-effective production of a variety of recombinant proteins, vaccine candidates, therapeutic proteins, enzymes, antibodies etc. [15][16][17][18][19][20][21][22] . Recently, a number of flexible approaches have been developed, which enabled the successfully production of pharmaceutically important complex proteins including human proteins and enzymes in plants 15,[19][20][21] .…”

Construct design, production, and characterization of Plasmodium falciparum 48/45 R0.6C subunit protein produced in Lactococcus lactis as candidate vaccine

“…Proper folding of cysteine-rich Pf s48/45 depends on correct formation of disulfide bridges. Since eukaryotic cells possess a sophisticated machinery for disulfide bond formation, recombinant Pf s48/45 has been produced in a range of Baculovirus ( Spodoptera frugiperda Sf9) cells [9], Vaccinia virus [10], Saccharomyces cerevisiae [11], and Pichia pastoris [11], Chlamydomonas reinhardtii [12], and Nicotiana benthamiana [13]. However, protein yields were rather low and in those cases where expression was successful, there was limited reactivity with mAbs against conformational TB epitopes suggesting misfolding.…”

When plant virology met Agrobacterium: the rise of the deconstructed clones

“…The CPMV-HT system relies solely on translational efficiency as opposed to replication, while tobravirus-based vectors are ideal for gene silencing and protein expression in roots. While this review has focussed on deconstructed vectors designed from the most commonly used viral taxons, it is important to note that there has been some work aimed at developing deconstructed vectors based other viruses, such as the cucumovirus cucumber mosaic virus (CMV; Fujiki et al, 2008) and the closterovirus beet yellows virus (BYV; Prokhnevsky et al, 2015).…”