Transient expression of Human papillomavirus type 16 L1 protein in Nicotiana benthamiana using an infectious tobamovirus vector

A Tobacco mosaic virus (TMV)-derived vector was used to express a native Human papillomavirus type 16 (HPV-16) L1 gene in Nicotiana benthamiana by means of infectious in vitro RNA transcripts inoculated onto N. benthamiana plants. HPV-16 L1 protein expression was quantitated by enzyme-linked immunosorbent assays (ELISA) after concentration of the plant extract. We estimated that the L1 product yield was 20-37 μg/kg of fresh leaf material. The L1 protein in the concentrated extract was antigenically characterised using the neutralising and conformation-specific Mabs H16:V5 and H16:E70, which bound to the plant-produced protein. Particles observed by transmission electron microscopy were mainly capsomers but virus-like particles (VLPs) similar to those produced in other systems were also present. Immunisation of rabbits with the concentrated plant extract induced a weak immune response. This is the first report of the successful expression of an HPV L1 gene in plants using a plant virus vector. © 2006 Elsevier B.V. All rights reserved.

Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants

Background We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes. Results We give an example of expression of heme-thiolate P450 to demonstrate effectiveness of this system. We have also designed vectors that take advantage of a dual luciferase assay system to analyse promoter sequences or post-transcriptional regulation of gene expression. We have demonstrated their utility by co-expression of putative transcription factors and the promoter sequence of potential target genes and show how orthologous promoter sequences respond to these genes. Finally, we have constructed a vector that has allowed us to investigate design features of hairpin constructs related to their ability to initiate RNA silencing, and have used these tools to study cis-regulatory effect of intron-containing gene constructs. Conclusion In developing a series of vectors ideally suited to transient expression analysis we have provided a resource that further advances the application of this technology. These minimal vectors are ideally suited to conventional cloning methods and we have used them to demonstrate their flexibility to investigate enzyme activity, transcription regulation and post-transcriptional regulatory processes in transient assays.

Transient expression of lacZ in particle bombarded Gracilaria changii (Gracilariales, Rhodophyta)

Using a Biolistic PDS 1000/He system, healthy thalli of Gracilaria changii were bombarded with gold particles coated with plasmid DNA containing the lacZ reporter gene. Transient expression of lacZ was observed in bombarded thalli under the rupture-disc pressures of 4482, 6206, 7584 and 8963 KPa, two days after bombardment. Although G. changii exhibits a slight blue background, positive expression and the background colour can be clearly differentiated. The results indicate that lacZ could be a useful reporter gene and that SV40 promoter could be an effective promoter for Gracilaria transformation.

Transient expression of lacZ in bombarded unicellular green alga Haematococcus pluvialis

This paper reports for the first time the transient expression of a reporter gene, LacZ, in the unicellular green alga Haematococcus pluvialis. By employing the micro-particle bombardment method, motile cells in the exponential phase showed transient expression of lacZ. This was detected in bombarded motile cells under the rupture-disc pressures of 3103 KPa and 4137 KPa. Transient expression of LacZ gene could not be observed in non-motile cells of this alga under the same transformation condition. No LacZ background was found in either the motile cells or the non-motile cells. The study suggests a promising potential of the SV40 promoter and the lacZ reporter gene in genetic engineering of unicellular green algae.

Changes in histone acetylation and methylation that are important for persistent but not transient expression of CCR4 in human CD4(+) T cells

Although regulation of CXCR3 and CCR4 is related to Th1 and Th2 differentiation, respectively, many CXCR3(+) and CCR4(+) cells do not express IFN-gamma and/or IL-4, suggesting that the chemokine receptor genes might be inducible by mechanisms that are lineage-independent. We investigated the regulation of CXCR3 versus IFNG, and CCR4 versus IL4 in human CD4(+) T cells by analyzing modifications of histone H3. In naive cord-blood cells, under nonpolarizing conditions not inducing IL4, CCR4 was induced to high levels without many of the activation-associated changes in promoter histone H3 found for both IL4 and CCR4 in Th2 cells. Importantly, CCR4 expression was stable in Th2 cells, but fell in nonpolarized cells after the cells were rested; this decline could be reversed by increasing histone acetylation using sodium butyrate. Patterns of histone H3 modifications in CXCR3(+) CCR4(-) and CXCR3(-) CCR4(+) CD4(+) T-cell subsets from adult blood matched those in cells cultured under polarizing conditions in vitro. Our data show that high-level lineage-independent induction of CCR4 can occur following T-cell activation without accessibility-associated changes in histone H3, but that without such changes expression is transient rather than persistent.

Use of microprojectile bombardment in transient expression assays to analyze protochlorophyllide reductase gene expression in greening maize seedling leaf cells

In young cells of leaf meristems the progenitors of chloroplasts are small organelles known as proplastids, which divide and differentiate into chloroplasts. However, in the absence of light, proplastids undergo a different sequence of development and become etioplasts. When light is supplied to etiolated plants during the "greening" process, etioplasts differentiate into chloroplasts containing chlorophyll. An important light dependent step in chlorophyll biosynthesis is the photoreduction of protochlorophyllide to chlorophyllide by the NADPH:protochlorophyllide reductase (PCR) enzyme. This enzyme is present at high activity only in etiolated tissue and during early stages of light-induced chlorophyll synthesis. The enzyme and its corresponding mRNAs decrease dramatically with prolonged exposure to light. We have investigated the light-dependent transcriptional regulation of a PCR gene in greening maize leaf cells using a transient expression assay based on microprojectile bombardment. The promoter region was isolated and cloned into a ?-glucuronidase (GUS) reporter gene expression plasmid. We have used this chimeric plasmid in tungsten particle bombardment of both etiolated and greening maize seedling leaves to determine whether the cloned promoter region contains regulatory sequences that control light-responsive PCR gene expression.

Transient expression of rabies virus G-glycoprotein using BHK-21 cells cultured in suspension

Objective To assess the expression of rabies virus G-glycoprotein (RVGP) expression using Semliki Forest virus as a vector in combination with BHK-21 cells cultured in suspension. Results A multilevel factorial design was used to quantify effects of temperature (33–37 C), fresh medium addition after the viral adsorption step (100–200 % with respect to the initial cell suspension volume before infection) and harvest time (8–40 h) on RVGP production. Experimental runs were performed in 24-well cell culture plates at a multiplicity of infection (MOI) of 16. An additional experiment in spinner-flask was performed at MOI of 9, using the optimal conditions determined in cell culture plates. Values for temperature, fresh medium addition and harvest time of 33 C, 100 % and 16 h, respectively, ensured the optimal RVGP production in culture plates. The volumetric yield (239 ng ml-1 ) in these conditions was higher than that reported previously for adherent cell culture. In spinner-flasks, the volumetric yield was improved (559 ng ml-1 ). Conclusion These results establish the basis for designing bioprocess to produce RVGP.

Inhibition of maize streak virus (MSV) replication by transient and transgenic expression of MSV replication-associated protein mutants

Maize streak disease is a severe agricultural problem in Africa and the development of maize genotypes resistant to the causal agent, Maize streak virus (MSV), is a priority. A transgenic approach to engineering MSV-resistant maize was developed and tested in this study. A pathogen-derived resistance strategy was adopted by using targeted deletions and nucleotide-substitution mutants of the multifunctional MSV replication-associated protein gene (rep). Various rep gene constructs were tested for their efficacy in limiting replication of wild-type MSV by co-bombardment of maize suspension cells together with an infectious genomic clone of MSV and assaying replicative forms of DNA by quantitative PCR. Digitaria sanguinalis, an MSV-sensitive grass species used as a model monocot, was then transformed with constructs that had inhibited virus replication in the transient-expression system. Challenge experiments using leafhopper-transmitted MSV indicated significant MSV resistance - from highly resistant to immune - in regenerated transgenic D. sanguinalis lines. Whereas regenerated lines containing a mutated full-length rep gene displayed developmental and growth defects, those containing a truncated rep gene both were fertile and displayed no growth defects, making the truncated gene a suitable candidate for the development of transgenic MSV-resistant maize. © 2007 SGM.

Transient gene expression in Medicago truncatula leaves via agroinfiltration

Transient expression is a powerful method for the functional characterization of genes. In this chapter, we outline a protocol for the transient expression of constructs in Medicago truncatula leaves using Agrobacterium tumefaciens infiltration. Using quantitative real-time PCR we demonstrate that the infiltration of a construct containing the LEGUME ANTHOCYANIN PRODUCTION 1 (LAP1) transcription factor results in the strong upregulation of key biosynthetic genes and the accumulation of anthocyanin pigment in the leaves after just 3 days. Thus, this method provides a rapid and powerful way to the discovery of downstream targets of M. truncatula transcription factors.

Development of a transient, high-level expression platform for protein production in plants

Plants are an attractive alternative to conventional expression systems for the production of recombinant proteins and useful biologics, however, the economic viability of plant made proteins is strongly yield dependent. This study aimed to improve transgene expression levels in the plant host Nicotiana benthamiana using the Agroinfiltration transient expression platform. Independent investigation of the physical, chemical and genetic features associated with Agroinfiltration identified factors that improved transformation frequencies, elevated transgene expression levels and ultimately improved protein yield. The major outcome of this research was a novel hyper-expression system for biofarming recombinant proteins in plants.

Engineering mRNA translation initiation to enhance transient gene expression in Chinese hamster ovary cells

To increase transient expression of recombinant proteins in Chinese hamster ovary cells, we have engineered their protein synthetic capacity by directed manipulation of mRNA translation initiation. To control this process we constructed a nonphosphorylatable Ser51Ala site-directed mutant of eIF2, a subunit of the trimeric eIF2 complex that is implicated in regulation of the global rate of mRNA translation initiation in eukaryotic cells. Phosphorylation of eIF2 by protein kinases inhibits eIF2 activity and is known to increase as cells perceive a range of stress conditions. Using single-and dual-gene plasmids introduced into CHO cells by electroporation, we found that transient expression of the eIF2 Ser51Ala mutant with firefly luciferase resulted in a 3-fold increase in reporter activity, relative to cells transfected with reporter only. This effect was maintained in transfected cells for at least 48 h after transfection. Expression of the wild-type eIF2 protein had no such effect. Elevated luciferase activity was associated with a reduction in the level of eIF2 phosphorylation in cells transfected with the mutant eIF2 construct. Transfection of CHO cells with the luciferase-only construct resulted in a marked decrease in the global rate of protein synthesis in the whole cell population 6 h post-transfection. However, expression of the mutant Ser51Ala or wild-type eIF2 proteins restored the rate of protein synthesis in transfected cells to a level equivalent to or exceeding that of control cells. Associated with this, entry of plasmid DNA into cells during electroporation was visualized by confocal microscopy using a rhodamine-labeled plasmid construct expressing green fluorescent protein. Six hours after transfection, plasmid DNA was present in all cells, albeit to a variable extent. These data suggest that entry of naked DNA into the cell itself functions to inhibit protein synthesis by signaling mechanisms affecting control of mRNA translation by eIF2. This work therefore forms the basis of a rational strategy to generically up-regulate transient expression of recombinant proteins by simultaneous host cell engineering.

Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: Comparison of the suitability of different HPV-16 L1 gene variants and different cell-compartment localization

Virus-like particle-based vaccines for high-risk human papillomaviruses (HPVs) appear to have great promise; however, cell culture-derived vaccines will probably be very expensive. The optimization of expression of different codon-optimized versions of the HPV-16 L1 capsid protein gene in plants has been explored by means of transient expression from a novel suite of Agrobacterium tumefaciens binary expression vectors, which allow targeting of recombinant protein to the cytoplasm, endoplasmic reticulum (ER) or chloroplasts. A gene resynthesized to reflect human codon usage expresses better than the native gene, which expresses better than a plant-optimized gene. Moreover, chloroplast localization allows significantly higher levels of accumulation of L1 protein than does cytoplasmic localization, whilst ER retention was least successful. High levels of L1 (>17% total soluble protein) could be produced via transient expression: the protein assembled into higher-order structures visible by electron microscopy, and a concentrated extract was highly immunogenic in mice after subcutaneous injection and elicited high-titre neutralizing antibodies. Transgenic tobacco plants expressing a human codon-optimized gene linked to a chloroplast-targeting signal expressed L1 at levels up to 11% of the total soluble protein. These are the highest levels of HPV L1 expression reported for plants: these results, and the excellent immunogenicity of the product, significantly improve the prospects of making a conventional HPV vaccine by this means. © 2007 SGM.

Expression of HIV-1 antigens in plants as potential subunit vaccines

Background Human immunodeficiency virus type 1 (HIV-1) has infected more than 40 million people worldwide, mainly in sub-Saharan Africa. The high prevalence of HIV-1 subtype C in southern Africa necessitates the development of cheap, effective vaccines. One means of production is the use of plants, for which a number of different techniques have been successfully developed. HIV-1 Pr55Gag is a promising HIV-1 vaccine candidate: we compared the expression of this and a truncated Gag (p17/p24) and the p24 capsid subunit in Nicotiana spp. using transgenic plants and transient expression via Agrobacterium tumefaciens and recombinant tobamovirus vectors. We also investigated the influence of subcellular localisation of recombinant protein to the chloroplast and the endoplasmic reticulum (ER) on protein yield. We partially purified a selected vaccine candidate and tested its stimulation of a humoral and cellular immune response in mice. Results Both transient and transgenic expression of the HIV antigens were successful, although expression of Pr55Gag was low in all systems; however, the Agrobacterium-mediated transient expression of p24 and p17/p24 yielded best, to more than 1 mg p24/kg fresh weight. Chloroplast targeted protein levels were highest in transient and transgenic expression of p24 and p17/p24. The transiently-expressed p17/p24 was not immunogenic in mice as a homologous vaccine, but it significantly boosted a humoral and T cell immune response primed by a gag DNA vaccine, pTHGagC. Conclusion Transient agroinfiltration was best for expression of all of the recombinant proteins tested, and p24 and p17/p24 were expressed at much higher levels than Pr55Gag. Our results highlight the usefulness of plastid signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The p17/p24 protein effectively boosted T cell and humoral responses in mice primed by the DNA vaccine pTHGagC, showing that this plant-produced protein has potential for use as a vaccine.

A rep-based hairpin inhibits replication of diverse maize streak virus isolates in a transient assay

Centre for High-Performance Computing, Rosebank, Cape Town, South Africa Maize streak disease, caused by the A strain of the African endemic geminivirus, maize streak mastrevirus (MSV-A), threatens the food security and livelihoods of subsistence farmers throughout sub-Saharan Africa. Using a well-established transient expression assay, this study investigated the potential of a spliceable-intron hairpin RNA (hpRNA) approach to interfere with MSV replication. Two strategies were explored: (i) an inverted repeat of a 662 bp region of the MSV replication-associated protein gene (rep), which is essential for virus replication and is therefore a good target for post-transcriptional gene silencing; and (ii) an inverted repeat of the viral long intergenic region (LIR), considered for its potential to trigger transcriptional silencing of the viral promoter region. After co-bombardment of cultured maize cells with each construct and an infectious partial dimer of the cognate virus genome (MSV-Kom), followed by viral replicativeform-specific PCR, it was clear that, whilst the hairpin rep construct (pHPrepDI662) completely inhibited MSV replication, the LIR hairpin construct was ineffective in this regard. In addition, pHPrepDI662 inhibited or reduced replication of six MSV-A genotypes representing the entire breadth of known MSV-A diversity. Further investigation by real-time PCR revealed that the pHPrepDI662 inverted repeat was 22-fold more effective at reducing virus replication than a construct containing the sense copy, whilst the antisense copy had no effect on replication when compared with the wild type. This is the first indication that an hpRNA strategy targeting MSV rep has the potential to protect transgenic. © 2011 SGM.

Heterologous promoter recognition leading to high-level expression of cloned foreign genes in Bombyx mori cell lines and larvae

The baculovirus expression system using the Autographa californica nuclear polyhedrosis virus (AcNPV) has been extensively utilized for high-level expression of cloned foreign genes, driven by the strong viral promoters of polyhedrin (polh) and p10 encoding genes. A parallel system using Bombyx mori nuclear polyhedrosis virus (BmNPV) is much less exploited because the choice and variety of BmNPV-based transfer vectors are limited. Using a transient expression assay, we have demonstrated here that the heterologous promoters of the very late genes polh and p10 from AcNPV function as efficiently in BmN cells as the BmNPV promoters. The location of the cloned foreign gene with respect to the promoter sequences was critical for achieving the highest levels of expression, following the order +35 > +1 > -3 > -8 nucleotides (nt) with respect to the polh or p10 start codons. We have successfully generated recombinant BmNPV harboring AcNPV promoters by homeologous recombination between AcNPV-based transfer vectors and BmNPV genomic DNA. Infection of BmN cell lines with recombinant BmNPV showed a temporal expression pattern, reaching very high levels in 60-72 h post infection. The recombinant BmNPV harboring the firefly luciferase-encoding gene under the control of AcNPV polh or p10 promoters, on infection of the silkworm larvae led to the synthesis of large quantities of luciferase. Such larvae emanated significant luminiscence instantaneously on administration of the substrate luciferin resulting in 'glowing silkworms'. The virus-infected larvae continued to glow for several hours and revealed the most abundant distribution of virus in the fat bodies. In larval expression also, the highest levels were achieved when the reporter gene was located at +35 nt of the polh.