Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana.

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing need in developing efficient methods for recombinant antibody production. As of 2019, there were more than 70 FDA-approved monoclonal antibodies, and there is exponential growth potential. Despite their promise, limiting factors for widespread use are manufacturing costs and complexity. Potentially, plants offer low-cost, safe, and easily scalable protein manufacturing strategies. Plants like Nicotiana benthamiana not only can correctly fold and assemble complex mammalian proteins but also can add critical post-translational modifications similar to those offered by mammalian cell cultures. In this work, by using native GFP and an acid-stable variant of green fluorescent protein (GFP) fused to human monoclonal antibodies, we were able to visualize the entire transient antibody expression and purification process from N. benthamiana plants. Depending on the experiment's purpose, native GFP fusion can ensure easier visualization during the expression phase in the plants, while acid-stable GFP fusion allows for visualization during downstream processing. This scalable and straightforward procedure can be performed by a single researcher to produce milligram quantities of highly pure antibody or antibody fusion proteins in a matter of days using only a few small plants. Such a technique can be extended to the visualization of any type of antibody purification process and potentially many other proteins, both in plant and other expression systems. Moreover, these techniques can benefit virtual instructions and be executed in a teaching laboratory by undergraduate students possessing minimal prior experience with molecular biology techniques, providing a foundation for project-based exploration with real-world applications.

A plant-derived cocaine hydrolase prevents cocaine overdose lethality and attenuates cocaine-induced drug seeking behavior.

Cocaine use disorders include short-term and acute pathologies (e.g. overdose) and long-term and chronic disorders (e.g. intractable addiction and post-abstinence relapse). There is currently no available treatment that can effectively reduce morbidity and mortality associated with cocaine overdose or that can effectively prevent relapse in recovering addicts. One recently developed approach to treat these problems is the use of enzymes that rapidly break down the active cocaine molecule into inactive metabolites. In particular, rational design and site-directed mutagenesis transformed human serum recombinant butyrylcholinesterase (BChE) into a highly efficient cocaine hydrolase with drastically improved catalytic efficiency toward (-)-cocaine. A current drawback preventing the clinical application of this promising enzyme-based therapy is the lack of a cost-effective production strategy that is also flexible enough to rapidly scale-up in response to continuous improvements in enzyme design. Plant-based expression systems provide a unique solution as this platform is designed for fast scalability, low cost and the advantage of performing eukaryotic protein modifications such as glycosylation. A Plant-derived form of the Cocaine Super Hydrolase (A199S/F227A/S287G/A328W/Y332G) we designate PCocSH protects mice from cocaine overdose, counters the lethal effects of acute cocaine overdose, and prevents reinstatement of extinguished drug-seeking behavior in mice that underwent place conditioning with cocaine. These results demonstrate that the novel PCocSH enzyme may well serve as an effective therapeutic for cocaine use disorders in a clinical setting.

A heterologous prime-boosting strategy with replicating Vaccinia virus vectors and plant-produced HIV-1 Gag/dgp41 virus-like particles.

Showing modest efficacy, the RV144 HIV-1 vaccine clinical trial utilized a non-replicating canarypox viral vector and a soluble gp120 protein boost. Here we built upon the RV144 strategy by developing a novel combination of a replicating, but highly-attenuated Vaccinia virus vector, NYVAC-KC, and plant-produced HIV-1 virus-like particles (VLPs). Both components contained the full-length Gag and a membrane anchored truncated gp41 presenting the membrane proximal external region with its conserved broadly neutralizing epitopes in the pre-fusion conformation. We tested different prime/boost combinations of these components in mice and showed that the group primed with NYVAC-KC and boosted with both the viral vectors and plant-produced VLPs have the most robust Gag-specific CD8 T cell responses, at 12.7% of CD8 T cells expressing IFN-γ in response to stimulation with five Gag epitopes. The same immunization group elicited the best systemic and mucosal antibody responses to Gag and dgp41 with a bias towards IgG1.

Immunological Characterization of Plant-Based HIV-1 Gag/Dgp41 Virus-Like Particles.

It is widely anticipated that a prophylactic vaccine may be needed to control the HIV/AIDS epidemic worldwide. Despite over two decades of research, a vaccine against HIV-1 remains elusive, although a recent clinical trial has shown promising results. Recent studies have focused on highly conserved domains within HIV-1 such as the membrane proximal external region (MPER) of the envelope glycoprotein, gp41. MPER has been shown to play critical roles in mucosal transmission of HIV-1, though this peptide is poorly immunogenic on its own. Here we provide evidence that plant-produced HIV-1 enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the MPER, transmembrane, and cytoplasmic domains (Dgp41) provides an effective platform to display MPER for use as an HIV vaccine candidate. Prime-boost strategies combining systemic and mucosal priming with systemic boosting using two different vaccine candidates (VLPs and CTB-MPR--a fusion of MPER and the B-subunit of cholera toxin) were investigated in BALB/c mice. Serum antibody responses against both the Gag and gp41 antigens were elicited when systemically primed with VLPs. These responses could be recalled following systemic boosting with VLPs. In addition, mucosal priming with VLPs allowed for a boosting response against Gag and gp41 when boosted with either candidate. Importantly, the VLPs also induced Gag-specific CD4 and CD8 T-cell responses. This report on the immunogenicity of plant-based Gag/Dgp41 VLPs may represent an important milestone on the road towards a broadly efficacious and inexpensive subunit vaccine against HIV-1.

Biophysical Characterization of a Vaccine Candidate against HIV-1: The Transmembrane and Membrane Proximal Domains of HIV-1 gp41 as a Maltose Binding Protein Fusion.

The membrane proximal region (MPR, residues 649-683) and transmembrane domain (TMD, residues 684-705) of the gp41 subunit of HIV-1's envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662-683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649-705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM). Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41.

Oligomerization status influences subcellular deposition and glycosylation of recombinant butyrylcholinesterase in Nicotiana benthamiana.

Plants have a proven track record for the expression of biopharmaceutically interesting proteins. Importantly, plants and mammals share a highly conserved secretory pathway that allows similar folding, assembly and posttranslational modifications of proteins. Human butyrylcholinesterase (BChE) is a highly sialylated, tetrameric serum protein, investigated as a bioscavenger for organophosphorous nerve agents. Expression of recombinant BChE (rBChE) in Nicotiana benthamiana results in accumulation of both monomers as well as assembled oligomers. In particular, we show here that co-expression of BChE with a novel gene-stacking vector, carrying six mammalian genes necessary for in planta protein sialylation, resulted in the generation of rBChE decorated with sialylated N-glycans. The N-glycosylation profile of monomeric rBChE secreted to the apoplast largely resembles the plasma-derived orthologue. In contrast, rBChE purified from total soluble protein extracts was decorated with a significant portion of ER-typical oligomannosidic structures. Biochemical analyses and live-cell imaging experiments indicated that impaired N-glycan processing is due to aberrant deposition of rBChE oligomers in the endoplasmic reticulum or endoplasmic-reticulum-derived compartments. In summary, we show the assembly of rBChE multimers, however, also points to the need for in-depth studies to explain the unexpected subcellular targeting of oligomeric BChE in plants.

Expression of human butyrylcholinesterase with an engineered glycosylation profile resembling the plasma-derived orthologue.

Human butyrylcholinesterase (BChE) is considered a candidate bioscavenger of nerve agents for use in pre- and post-exposure treatment. However, the presence and functional necessity of complex N-glycans (i.e. sialylated structures) is a challenging issue in respect to its recombinant expression. Here we transiently co-expressed BChE cDNA in the model plant Nicotiana benthamiana with vectors carrying the genes necessary for in planta protein sialylation. Site-specific sugar profiling of secreted recombinant BChE (rBChE) collected from the intercellular fluid revealed the presence of mono- and di-sialylated N-glycans, which largely resembles to the plasma-derived orthologue. Attempts to increase that sialylation content of rBChE by the over-expression of an additional glycosylation enzyme that generates branched N-glycans (i.e. ß1,4-N-acetylglucosaminyl-transferase IV), allowed the production of rBChE decorated with tri-sialylated structures (up to 70%). Sialylated and non-sialylated plant-derived rBChE exhibited functional in vitro activity comparable to that of its commercially available equine-derived counterpart. These results demonstrate the ability of plants to generate valuable proteins with designed sialylated glycosylation profiles optimized for therapeutic efficacy. Moreover, the efficient synthesis of carbohydrates present only in minute amounts on the native protein (tri-sialylated N-glycans) facilitates the generation of a product with superior efficacies and/or new therapeutic functions.

Meeting report VLPNPV: Session 5: Plant based technology.

The VLPNPV 2014 Conference that was convened at the Salk institute was the second conference of its kind to focus on advances in production, purification, and delivery of virus-like particles (VLPs) and nanoparticles. Many exciting developments were reported and discussed in this interdisciplinary arena, but here we report specifically on the contributions of plant-based platforms to VLP vaccine technology as reported in the section of the conference devoted to the topic as well in additional presentations throughout the meeting. The increasing popularity of plant production platforms is due to their lower cost, scalability, and lack of contaminating animal pathogens seen with other systems. Reports include production of complex VLPs consisting of 4 proteins expressed at finely-tuned expression levels, a prime-boost strategy for HIV vaccination using plant-made VLPs and a live viral vector, and the characterization and development of plant viral nanoparticles for use in cancer vaccines, drug delivery, and bioimaging.

Nicotinic stimulation induces Tristetraprolin over-production and attenuates inflammation in muscle.

Cholinergic signaling suppresses inflammation in blood and brain and attenuates apoptosis in other tissues, but whether it blocks inflammation in skeletal muscle under toxicant exposure, injuries and diseases remained unexplored. Here, we report nicotinic attenuation of inflammation and alteration of apoptotic protein expression pattern in murine muscle tissue and cultured myotubes, involving the RNA-binding protein, Tristetraprolin, and the anti-apoptotic protein, Mcl-1. In muscles and C2C12 myotubes, cholinergic excitation by exposure to nicotine or the organophosphorous pesticide, Paraoxon, induced Tristetraprolin overproduction while reducing pro-inflammatory transcripts such as IL-6, CXCL1 (KC) and CCL2 (MCP-1). Furthermore, nicotinic excitation under exposure to the bacterial endotoxin LPS attenuated over-expression of the CCL2 and suppressed the transcriptional activity of NF-ĸB and AP-1. Tristetraprolin was essential for this anti-inflammatory effect of nicotine in basal conditions. However, its knockdown also impaired the pro-inflammatory response to LPS. Finally, in vivo administration of Paraoxon or recombinant Acetylcholinesterase, leading respectively to either gain or loss of cholinergic signaling, modified muscle expression of key mRNA processing factors and several of their apoptosis-related targets. Specifically, cholinergic imbalances enhanced the kinase activators of the Serine-Arginine splicing kinases, Clk1 and Clk3. Moreover, Paraoxon raised the levels of the anti-apoptotic protein, Mcl-1, through a previously unrecognized polyadenylation site selection mechanism, producing longer, less stable Mcl-1 mRNA transcripts. Together, our findings demonstrate that in addition to activating muscle function, acetylcholine regulates muscle inflammation and cell survival, and point to Tristetraprolin and the choice of Mcl-1 mRNA polyadenylation sites as potential key players in muscle reactions to insults.

Plant-derived human butyrylcholinesterase, but not an organophosphorous-compound hydrolyzing variant thereof, protects rodents against nerve agents.

The concept of using cholinesterase bioscavengers for prophylaxis against organophosphorous nerve agents and pesticides has progressed from the bench to clinical trial. However, the supply of the native human proteins is either limited (e.g., plasma-derived butyrylcholinesterase and erythrocytic acetylcholinesterase) or nonexisting (synaptic acetylcholinesterase). Here we identify a unique form of recombinant human butyrylcholinesterase that mimics the native enzyme assembly into tetramers; this form provides extended effective pharmacokinetics that is significantly enhanced by polyethylene glycol conjugation. We further demonstrate that this enzyme (but not a G117H/E197Q organophosphorus acid anhydride hydrolase catalytic variant) can prevent morbidity and mortality associated with organophosphorous nerve agent and pesticide exposure of animal subjects of two model species.

Biochemical and immunological characterization of the plant-derived candidate human immunodeficiency virus type 1 mucosal vaccine CTB-MPR.

Plants are potentially the most economical platforms for the large-scale production of recombinant proteins. Thus, plant-based expression of subunit human immunodeficiency virus type 1 (HIV-1) vaccines provides an opportunity for their global use against the acquired immunodeficiency syndrome pandemic. CTB-MPR(649-684)[CTB, cholera toxin B subunit; MPR, membrane proximal (ectodomain) region of gp41] is an HIV-1 vaccine candidate that has been shown previously to induce antibodies that block a pathway of HIV-1 mucosal transmission. In this article, the molecular characterization of CTB-MPR(649-684) expressed in transgenic Nicotiana benthamiana plants is reported. Virtually all of the CTB-MPR(649-684) proteins expressed in the selected line were shown to have assembled into pentameric, GM1 ganglioside-binding complexes. Detailed biochemical analyses on the purified protein revealed that it was N-glycosylated, predominantly with high-mannose-type glycans (more than 75%), as predicted from a consensus asparagine-X-serine/threonine (Asn-X-Ser/Thr) N-glycosylation sequon on the CTB domain and an endoplasmic reticulum retention signal attached at the C-terminus of the fusion protein. Despite this modification, the plant-expressed protein retained the nanomolar affinity to GM1 ganglioside and the critical antigenicity of the MPR(649-684) moiety. Furthermore, the protein induced mucosal and serum anti-MPR(649-684) antibodies in mice after mucosal prime-systemic boost immunization. Our data indicate that plant-based expression can be a viable alternative for the production of this subunit HIV-1 vaccine candidate.

Hairy-root organ cultures for the production of human acetylcholinesterase.

BACKGROUND: Human cholinesterases can be used as a bioscavenger of organophosphate toxins used as pesticides and chemical warfare nerve agents. The practicality of this approach depends on the availability of the human enzymes, but because of inherent supply and regulatory constraints, a suitable production system is yet to be identified. RESULTS: As a promising alternative, we report the creation of "hairy root" organ cultures derived via Agrobacterium rhizogenes-mediated transformation from human acetylcholinesterase-expressing transgenic Nicotiana benthamiana plants. Acetylcholinesterase-expressing hairy root cultures had a slower growth rate, reached to the stationary phase faster and grew to lower maximal densities as compared to wild type control cultures. Acetylcholinesterase accumulated to levels of up to 3.3% of total soluble protein, ~3 fold higher than the expression level observed in the parental plant. The enzyme was purified to electrophoretic homogeneity. Enzymatic properties were nearly identical to those of the transgenic plant-derived enzyme as well as to those of mammalian cell culture derived enzyme. Pharmacokinetic properties of the hairy-root culture derived enzyme demonstrated a biphasic clearing profile. We demonstrate that master banking of plant material is possible by storage at 4 degrees C for up to 5 months. CONCLUSION: Our results support the feasibility of using plant organ cultures as a successful alternative to traditional transgenic plant and mammalian cell culture technologies.

Increased organophosphate scavenging in a butyrylcholinesterase mutant.

Nicotiana benthamiana plant lines expressing a reengineered human butyrylcholinesterase (BChE) with enhanced cocaine hydrolase activity were created. Subsequent purification and biochemical analysis revealed that compared to wild-type butyrylcholinesterase, the cocaine hydrolase displayed increased affinity to the organophosphate (OP) pesticides paraoxon (6.8 4x 10(-10)M vs. 1.11 x 10(-8)M) and malaoxon (9.81 x 10(-8)M vs. 5.99 x 10(-7)M). Furthermore, the cocaine hydrolase retained identical anticholinesterase binding profiles for all other compounds tested. Thus we have demonstrated a potential large-scale production platform for a multivalent antidote for cocaine and anticholinesterase poisoning.

Changes in readthrough acetylcholinesterase expression modulate amyloid-beta pathology.

Alzheimer's disease has long been known to involve cholinergic deficits, but the linkage between cholinergic gene expression and the Alzheimer's disease amyloid pathology has remained incompletely understood. One known link involves synaptic acetylcholinesterase (AChE-S), shown to accelerate amyloid fibrils formation. Here, we report that the 'Readthrough' AChE-R splice variant, which differs from AChE-S in its 26 C-terminal residues, inversely exerts neuroprotective effects from amyloid beta (Abeta) induced toxicity. In vitro, highly purified AChE-R dose-dependently suppressed the formation of insoluble Abeta oligomers and fibrils and abolished Abeta toxicity to cultured cells, competing with the prevalent AChE-S protein which facilitates these processes. In vivo, double transgenic APPsw/AChE-R mice showed lower plaque burden, fewer reactive astrocytes and less dendritic damage than single APPsw mice, inverse to reported acceleration of these features in double APPsw/AChE-S mice. In hippocampi from Alzheimer's disease patients (n = 10), dentate gyrus neurons showed significantly elevated AChE-R mRNA and reduced AChE-S mRNA. However, immunoblot analyses revealed drastic reductions in the levels of intact AChE-R protein, suggesting that its selective loss in the Alzheimer's disease brain exacerbates the Abeta-induced damages and revealing a previously unforeseen linkage between cholinergic and amyloidogenic events.

Translational control of recombinant human acetylcholinesterase accumulation in plants.

BACKGROUND: Codon usage differences are known to regulate the levels of gene expression in a species-specific manner, with the primary factors often cited to be mRNA processing and accumulation. We have challenged this conclusion by expressing the human acetylcholinesterase coding sequence in transgenic plants in its native GC-rich sequence and compared to a matched sequence with (dicotyledonous) plant-optimized codon usage and a lower GC content. RESULTS: We demonstrate a 5 to 10 fold increase in accumulation levels of the "synaptic" splice variant of human acetylcholinesterase in Nicotiana benthamiana plants expressing the optimized gene as compared to the native human sequence. Both transient expression assays and stable transformants demonstrated conspicuously increased accumulation levels. Importantly, we find that the increase is not a result of increased levels of acetylcholinesterase mRNA, but rather its facilitated translation, possibly due to the reduced energy required to unfold the sequence-optimized mRNA. CONCLUSION: Our findings demonstrate that codon usage differences may regulate gene expression at different levels and anticipate translational control of acetylcholinesterase gene expression in its native mammalian host as well.

Plant-derived human acetylcholinesterase-R provides protection from lethal organophosphate poisoning and its chronic aftermath.

Therapeutically valuable proteins are often rare and/or unstable in their natural context, calling for production solutions in heterologous systems. A relevant example is that of the stress-induced, normally rare, and naturally unstable "read-through" human acetylcholinesterase variant, AChE-R. AChE-R shares its active site with the synaptic AChE-S variant, which is the target of poisonous organophosphate anticholinesterase insecticides such as the parathion metabolite paraoxon. Inherent AChE-R overproduction under organophosphate intoxication confers both short-term protection (as a bioscavenger) and long-term neuromuscular damages (as a regulator). Here we report the purification, characterization, and testing of human, endoplasmic reticulum-retained AChE-R(ER) produced from plant-optimized cDNA in Nicotiana benthamiana plants. AChE-R(ER) purified to homogeneity showed indistinguishable biochemical properties, with IC50 = 10(-7) M for the organophosphate paraoxon, similar to mammalian cell culture-derived AChE. In vivo titration showed dose-dependent protection by intravenously injected AChE-R(ER) of FVB/N male mice challenged with a lethal dose of paraoxon, with complete elimination of short-term clinical symptoms at near molar equivalence. By 10 days postexposure, AChE-R prophylaxis markedly limited postexposure increases in plasma murine AChE-R levels while minimizing the organophosphate-induced neuromuscular junction dismorphology. Our findings present plant-produced AChE-R(ER) as a bimodal agent, conferring both short- and long-term protection from organophosphate intoxication.

Adaptive changes in acetylcholinesterase gene expression as mediators of recovery from chemical and biological insults.

Both organophosphate (OP) exposure and bacterial infection notably induce short- and long-term cholinergic responses. These span the central and peripheral nervous system, neuromuscular pathway and hematopoietic cells and involve over-expression of the "readthrough" variant of acetylcholinesterase, AChE-R, and its naturally cleavable C-terminal peptide ARP. However, the causal involvement of these changes with post-exposure recovery as opposed to apoptotic events remained to be demonstrated. Here, we report the establishment of stably transfected cell lines expressing catalytically active human "synaptic" AChE-S or AChE-R which are fully viable and non-apoptotic. In addition, intraperitoneally injected synthetic mouse ARP (mARP) elevated serum AChE levels post-paraoxon exposure. Moreover, mARP treatment ameliorated post-exposure increases in corticosterone and decreases in AChE gene expression and facilitated earlier retrieval of motor activity following both paraoxon and lipopolysaccharide (LPS) exposures. Our findings suggest a potential physiological role for overproduction of AChE-R and the ARP peptide following exposure to both chemical warfare agents and bacterial LPS.

Purification of transgenic plant-derived recombinant human acetylcholinesterase-R.

Nicotiana benthamiana plants were engineered to express a codon-optimized gene encoding the human acetylcholinesterase-R (AChE) isoform. The transgenic plants expressed the protein at >0.4% of total soluble protein, and the plant-produced enzyme was purified to homogeneity. Following lysis, procainamide affinity chromatography and anion-exchange chromatography, more than 400-fold purification was achieved and electrophoretic purity was obtained. This pure protein is kinetically indistinguishable from the only commercially available source of human acetylcholinesterase, which is produced in mammalian cell culture. Thus, we have demonstrated a model system for the production of acetylcholinesterase, which is not susceptible to the quantitative limitations or mammalian pathogens associated with purification from mammalian cell culture or human serum.

A mucosally targeted subunit vaccine candidate eliciting HIV-1 transcytosis-blocking Abs.

A vaccine that would engage the mucosal immune system against a broad range of HIV-1 subtypes and prevent epithelial transmission is highly desirable. Here we report fusing the mucosal targeting B subunit of cholera toxin to the conserved galactosylceramide-binding domain (including the ELDKWA-neutralizing epitope) of the HIV-1 gp41 envelope protein, which mediates the transcytosis of HIV-1 across the mucosal epithelia. Chimeric protein expressed in bacteria or plants assembled into oligomers that were capable of binding galactosyl-ceramide and G(M1) gangliosides. Mucosal (intranasal) administration in mice of the purified chimeric protein followed by an i.p. boost resulted in transcytosis-neutralizing serum IgG and mucosal IgA responses and induced immunological memory. Plant production of mucosally targeted immunogens could be particularly useful for immunization programs in developing countries, where desirable product traits include low cost of manufacture, heat stability, and needle-free delivery.

Geminivirus vectors for high-level expression of foreign proteins in plant cells.

Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins.