Bean Yellow Dwarf Virus replicons for high-level transgene expression in transgenic plants and cell cultures

A novel stable transgenic plant expression system was developed using elements of the replication machinery of Bean Yellow Dwarf Virus (BeYDV). The system contains two transgenes: 1) The BeYDV replicon vector with an expression cassette flanked by cis-acting DNA elements of BeYDV, and 2) The viral replication initiator protein (Rep) controlled by an alcohol-inducible promoter. When Rep expression was triggered by treatment with ethanol, it induced release of the BeYDV replicon from stably integrated T-DNA and episomal replication to high copy number. Replicon amplification resulted in substantially increased transgene mRNA levels (up to 80-fold) and translation products (up to 10-fold) after induction of Rep expression by ethanol treatment in tobacco NT1 cells and leaves of whole potato plants. Thus, the BeYDV stable transformant replicon system is a powerful tool for plant-based production of recombinant proteins.     

Multi-tasking of nonstructural gene products is required for bean yellow dwarf geminivirus transcriptional regulation

Mastreviridae, of the family geminiviridae, possess a monopartite genome and are transmitted by leafhoppers. Bean yellow dwarf dirus (BeYDV) is a mastrevirus which originated from South Africa and infects dicoyledenous plants, a feature unusual for mastreviridae. Previously, the nonstructural proteins Rep and RepA were examined with respect to their independent roles in BeYDV replication. This was achieved by placing both gene products under independent constitutive promoter control and examining their effects on replication-competent constructs. In the current study, Rep and RepA are examined further for their roles in regulating BeYDV gene expression using a series of replication-incompetent constructs. While both Rep and RepA are found to behave as equally potent inhibitors of complementary-sense gene expression, they differ considerably with respect to their abilities to transactivate virion-sense gene expression. Furthermore, RepA is identified as playing more than one role in this transactivation process. A nuclear localization domain is identified in Rep which is absent in RepA, and Rep-RepA interactions are examined under in vivo conditions. The study concludes with an investigation into the expression strategies of the BeYDV capsid protein.     

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.     

High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle 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.     

A DNA replicon system for rapid high‐level production of virus‐like particles in plants

Recombinant virus‐like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low‐level antigen accumulation and long‐time frame to produce transgenic plants are the two major roadblocks in the practical development of plant‐based VLP production. In this article, we describe the optimization of geminivirus‐derived DNA replicon vectors for rapid, high‐yield plant‐based production of VLPs. Co‐delivery of bean yellow dwarf virus (BeYDV)‐derived vector and Rep/RepA‐supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 5 days. Co‐expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built‐in Rep/RepA cassette without P19 drove protein expression at similar levels as the three‐component system. These results demonstrate the advantages of fast and high‐level production of VLP‐based vaccines using the BeYDV‐derived DNA replicon system for transient expression in plants. Biotechnol. Bioeng. 2009;103: 706–714. © 2009 Wiley Periodicals, Inc.     

Production of recombinant proteins in clonal root cultures using episomal expression vectors

We have developed a fully contained system for expressing recombinant proteins that is based on clonal root cultures and episomal expression vectors. Clonal root lines expressing green fluorescent protein (GFP) or human growth hormone were generated from Nicotiana benthamiana leaves infected with the tobacco mosaic virus-based vector 30B after exposure to Agrobacterium rhizogenes. These lines accumulated GFP at over 50 mg per kg fresh tissue, a level that is comparable with other plant production systems in early stage development. Accumulation of both hGH and GFP in the clonal root lines was sustained over a 3-year period, and in the absence of antibiotic selection. This technology shows promise for commercial production of vaccine antigens and therapeutic proteins in contained facilities.     

Glyco-engineering of biotherapeutic proteins in plants

Many therapeutic glycoproteins have been successfully generated in plants. Plants have advantages regarding practical and economic concerns, and safety of protein production over other existing systems. However, plants are not ideal expression systems for the production of biopharmaceutical proteins, due to the fact that they are incapable of the authentic human N-glycosylation process. The majority of therapeutic proteins are glycoproteins which harbor N-glycans, which are often essential for their stability, folding, and biological activity. Thus, several glyco-engineering strategies have emerged for the tailor-making of N-glycosylation in plants, including glycoprotein subcellular targeting, the inhibition of plant specific glycosyltranferases, or the addition of human specific glycosyltransferases. This article focuses on plant N-glycosylation structure, glycosylation variation in plant cell, plant expression system of glycoproteins, and impact of glycosylation on immunological function. Furthermore, plant glyco-engineering techniques currently being developed to overcome the limitations of plant expression systems in the production of therapeutic glycoproteins will be discussed in this review.     

Purification of replication factors using insect and mammalian cell expression systems

Purification of factors for DNA replication in an amount sufficient for detailed biochemical characterization is essential to elucidating its mechanisms. Insect cell expression systems are commonly used for purification of the factors proven to be difficult to deal with in bacteria. We describe first the detailed protocols for purification of mammalian Mcm complexes including the Mcm2/3/4/5/6/7 heterohexamer expressed in insect cells. We then describe a convenient and economical system in which large-sized proteins and multi-factor complexes can be transiently overexpressed in human 293T cells and be rapidly purified in a large quantity. We describe various expression vectors and detailed methods for transfection and purification of various replication factors which have been difficult to obtain in a sufficient amount in other systems. Availability of efficient methods to overproduce and purify the proteins that have been challenging would facilitate the enzymatic analyses of the processes of DNA replication.     

Use of viral replicons for the expression of genes in plants

Autonomously replicating virus-based vectors have been investigated as a means of introducing heterologous genes into plants. This approach has a number of potential advantages over stable genetic transformation, particularly in terms of speed and levels of expression that can be obtained. Several groups of plant viruses, with genomes consisting of both DNA and RNA, have been investigated as possible gene vectors. In the case of DNA viruses, it has generally been possible to identify nonessential regions of the genome that can be replaced by foreign sequences. However, there appear to be limitations on the size of insert which can be tolerated. In the case of RNA viruses, replacement of viral sequences usually has a drastic effect on the viability. However, in several cases it has proved possible to substantially increase the size of the viral genome by the direct insertion of additional sequences while still retaining the ability of the viruses to multiply and spread in plants. These RNA virus-based systems appear to have the greatest potential as gene vectors.     

In planta protein-protein interactions assessed using a nanovirus-based replication and expression system

The multipartite genome of the nanovirus Faba bean necrotic yellows virus, which consists of one gene on each DNA component, was exploited to construct a series of virus-based episomal vectors designed for transient replication and gene expression in plants. This nanovirus based expression system yields high levels of protein which allows isolation of recombinant protein and protein complexes from plant tissues. As examples, we demonstrated in planta interaction between the nanovirus F-box protein Clink and SKP1, a constituent of the ubiquitin-dependent protein turnover pathway. Thus, replicative nanovirus vectors provide a simple and efficient means for in planta characterization of protein-protein interaction.     

Plant virus expression systems for transient production of recombinant allergens in Nicotiana benthamiana

In recent years, several studies have demonstrated the use of autonomously replicating plant viruses as vehicles to express a variety of therapeutic molecules of pharmaceutical interest. Plant virus vectors for expression of heterologous proteins in plants represent an attractive biotechnological tool to complement the conventional production of recombinant proteins in bacterial, fungal, or mammalian cells. Virus vectors are advantageous when high levels of gene expression are desired within a short time, although the instability of the foreign genes in the viral genome may present problems. Similar levels of foreign protein production in transgenic plants often are unattainable, in some cases because of the toxicity of the foreign protein. Now virus-based vectors are for the first time investigated as a means of producing recombinant allergens in plants. Several plant virus vectors have been developed for the expression of foreign proteins. Here, we describe the utilization of tobacco mosaic virus- and potato virus X-based vectors for the transient expression of plant allergens in Nicotiana benthamiana plants. One approach involves the inoculation of tobacco plants with infectious RNA transcribed in vitro from a cDNA copy of the recombinant viral genome. Another approach utilizes the transfection of whole plants from wounds inoculated with Agrobacterium tumefaciens containing cDNA copies of recombinant plus-sense RNA viruses.     

Microfabricated reaction and separation systems

Over the past year there have been a number of recent advances in the fields of miniaturized reaction and separation systems, including the construction of fully integrated 'lab-on-a-chip' systems. Microreactors, which initially targeted DNA-based reactions such as the polymerase chain reaction, are now used in several other chemical and biochemical assays. Miniaturized separation columns are currently employed for analyzing a wide variety of samples including DNA, RNA, proteins and cells. Although significant advances have been made at the component level, the realization of an integrated analysis system still remains at the early stages of development.     

Affinity chromatography system for parallel purification of recombinant protein samples

In protein engineering, the tasks of generating and testing a large number of variants of a molecule and of optimizing expression conditions for one distinct molecule create the need for purification methods that can handle a large number of samples simultaneously. We describe the development and some application results of a simple affinity chromatography system that can be used for the parallel purification of 24 protein samples, yielding sufficient quantities for biochemical and functional analysis. Advantages of this system over existing systems are as follows. Compared with commercially available complete chromatography systems, the costs of this system are minimal. In comparison with vacuum systems with various outlets, and with batch purification systems where centrifugation is necessary, this system allows gentler processing of the samples. This could be important for proteins that are easily damaged.     

Comparison between gene expression of conidia and germinating phase in Trichophyton rubrum

Trichophyton rubrum is a dominating superficial dermatophyte, whose conidial germination is correlated to pathopoiesis and a highly important developmental process. To investigate the changes of physiology, biochemistry and cytology during the germination, we selected 3364 function identified ESTs from T. rubrum cDNA library to construct cDNA microarrays, and compared the gene expression levels of conidia and germinating phase. Data analysis indicated that 335 genes were up-regulated during the germination, which mainly encoded translated, modified proteins and structural proteins.The constituents of cell wall and cell membrane were synthetized abundantly, suggesting that they are the foundation of cell morphogenesis. The ingredients of the two-component signal transduction system were up-regulated, presuming that they were important for the conidial germination. Genes of various metabolic pathways were expressed prosperously, especially the genes that participated in glycolysis and oxidative phosphorylation were up-regulated on the whole, demonstrating that in the environment with sufficient oxygen and glucose, conidia obtained energy through aerobic respiration.This paper provides important clues which are helpful to understanding the changes in gene expression, signal conduction and metabolism characteristics during T. rubrum conidial germination, and possess significant meaning to the study of other superficial dermatophytes.