Improved expression of recombinant plant-made hEGF

Key message

The yield of recombinant hEGF was increased approximately tenfold through a range of optimisations. Further, the recombinant protein was found to have biological activity comparable to commercial hEGF.

Abstract

Human epidermal growth factor (hEGF) is a powerful mitogen that can enhance the healing of a wide range of injuries, including burns, cuts, diabetic ulcers and gastric ulcers. However, despite its clinical value, hEGF is only consistently used for the treatment of chronic diabetic ulcers due to its high cost. In this study, hEGF was transiently expressed in Nicotiana benthamiana plants and targeted to the apoplast, ER and vacuole. Several other approaches were also included in a stepwise fashion to identify the optimal conditions for the expression of recombinant hEGF. Expression was found to be highest in the vacuole, while targeting hEGF to the ER caused a decrease in total soluble protein (TSP). Using a codon optimised sequence was found to increase vacuolar targeted hEGF yield by ~34 %, while it was unable to increase the yield of ER targeted hEGF. The use of the P19 silencing inhibitor was able to further increase expression by over threefold, and using 5-week-old plants significantly increased expression compared to 4- or 6-week-old-plants. The combined effect of these optimisations increased expression tenfold over the initial apoplast targeted construct to an average yield of 6.24 % of TSP. The plant-made hEGF was then shown to be equivalent to commercial E. coli derived hEGF in its ability to promote the proliferation of mouse keratinocytes. This study supports the potential for plants to be used for the commercial production of hEGF, and identifies a potential limitation for the further improvement of recombinant protein yields.     

Expression and characteristic of synthetic human epidermal growth factor (hEGF) in transgenic tobacco plants

To improve the accumulation of recombinant human epidermal growth factor (hEGF) in transgenic tobacco, a highly effective vector was constructed and transformed via Agrobacterium tumefaciens. The hEGF content in transgenic tobacco was up to 0.3% of the total soluble protein. Using the Vero E6 cell expansion assay and the MTT method for cell proliferation, hEGF produced by transgenic tobacco significantly stimulated Vero E6 cell expansion and proliferation, the same as commercial hEGF products.     

Influence of cytoplasmic heat shock protein 70 on viral infection of Nicotiana benthamiana

The accumulation of heat shock protein 70 (Hsp70) generally occurs in plants infected with viruses. However, the effect of Hsp70 accumulation on plant viral infection and pathogenesis remains elusive. In this study, the expression of six Hsp70 genes was found to be induced by the four diverse RNA viruses, Tobacco mosaic virus, Potato virus X (PVX), Cucumber mosaic virus and Watermelon mosaic virus, in Nicotiana benthamiana. Heat treatment enhanced the accumulation and systemic infection of these viruses. Similar results were obtained for viral infection in plants heterologously expressing an Arabidopsis cytoplasmic Hsp70 through either a PVX vector or Agrobacterium infiltration. In contrast, viral infection was compromised in cytoplasmic NbHsp70c‐1 gene‐silenced plants. These data demonstrate that the cytoplasmic Hsp70s can enhance the infection of N. benthamiana by diverse viruses.     

Accumulation of human EGF in nectar of transformed plants of Nicotiana langsdorffii x N. sanderae and transfer to honey by bees

Honey has been used successfully in wound healing for thousands of years. The peptide hormone human epidermal growth factor (hEGF) is also known to have a beneficial effect in various wound healing processes via mechanisms that differ from those for honey. In this study, we show that hEGF can be incorporated into honey via nectar. Plants of Nicotiana langsdorffii × N. sanderae were transformed with the gene for hEGF, equipped with a nectary‐targeted promoter and a signal sequence for secretion to nectar. These plants accumulated hEGF in the nectar. The maximum hEGF concentration recorded with ELISA in these plants is 2.5 ng·ml^?1. There is a significant linear relationship (P < 0.001) between hEGF concentration and induction of hEGF‐receptor phosphorylation. Since the flower morphology of these plants did not allow production of honey from their nectar, we used feeding solutions, spiked with synthetic hEGF, to study transfer of this peptide into honey through bee activity. Transfer of hEGF from a feeding solution to honey by bees occurred with retention of the hEGF concentration and the capacity to induce hEGF‐receptor phosphorylation. These observations indicate that plants can function as a production platform for honey containing biologically active peptides, which may enhance wound healing and other biological processes.     

Molecular farming of human tissue transglutaminase in tobacco plants

In this study we have utilized Nicotiana tabacum with a molecular farming purpose in attempt of producing transgenic plants expressing the human tissue transglutaminase (htTG). Three plant expression constructs were used enabling targeting and accumulation of the recombinant protein into the plant cell cytosol (cyto), the chloroplasts (chl) and the apoplastic space (apo). Analysis of transgenic T₀ plants revealed that recombinant htTG was detectable in all three transgenic lines and the accumulation levels were in a range of 18–75 μg/g of leaf material. In the T₁ generation, the recombinant htTG was still expressed at high level and a significant catalytic activity was detected into the leaf protein extracts. Southern blot analyses revealed that apo and chl plants of T₁ generation possess a high copy number of the recombinant htTG in their genome, while the cyto plants carry a single copy.     

Expression of a human lactoferrin N-lobe in Nicotiana benthmiana with potato virus X-based agroinfection

A DNA fragment encoding the N-terminal half (N-lobe) of the human lactoferrin (hLfN) gene has now been cloned into recombinant Potexvirus potato virus X (PVX) vector and expressed in Nicotiana benthmiana using agroinfection. Western blot analysis showed the recombinant protein with an apparent molecular mass on electrophoresis of ca. 40 kDa, corresponding to the predicted size of the hLfN. The yield of hLfN reached a maximum (up to 0.6% of total soluble proteins) when recombinant PVX systemically infected an entire plant. Protein extracts from infected plants had antibacterial activity.     

Plant-produced recombinant influenza vaccine based on virus-like HBc particles carrying an extracellular domain of M2 protein

Conventional influenza vaccines are based on a virus obtained in chicken embryos or its components. The high variability of the surface proteins of influenza virus, hemagglutinin and neuraminidase, requires strain-specific vaccines matching the antigenic specificity of newly emerging virus strains to be developed. A recombinant vaccine based on a highly conservative influenza virus protein M2 fused to a nanosized carrier particle can be an attractive alternative to traditional vaccines. We have constructed a recombinant viral vector based on potato X virus that provides for expression in the Nicotiana benthamiana plants of a hybrid protein M2eHBc consisting of an extracellular domain of influenza virus M2 protein (M2e) fused to hepatitis B core antigen (HBc). This vector was introduced into plant cells by infiltrating leaves with agrobacteria carrying the viral vector. The hybrid protein M2eHBc was synthesized in the infected N. benthamiana plants in an amount reaching 1–2% of the total soluble protein and formed virus-like particles with the M2e peptide presented on the surface. Methods of isolation and purification of M2eHBc particles from plant producers were elaborated. Experiments on mice have shown a high immunogenicity of the plant-produced M2eHBc particles and their protective effect against lethal influenza challenge. The developed transient expression system can be used for production of M2e-based candidate influenza vaccine in plants.     

Bioactivity studies of Huh-7 cells derived human epidermal growth factor expressed in Pichia pastoris

Previously, we have reported cloning of human epidermal growth factor gene from Huh-7 cells and its extracellular expression in Pichia pastoris. The presented work is a detailed report regarding molecular characterization of Huh-7 cells-derived hEGF expressed in Pichia pastoris with special reference to its glycosylation profiling and bioactivity studies. Densitometric scanning of SDS-PAGE separated extracellular proteins from hEGF recombinant Pichia pastoris strain indicated that about 84% of the extracellular proteins were glycosylated. Size exclusion chromatography using Superdex 75 prep grade column was successfully utilized to separate fractions containing glycosylated and non-glycosylated extracellular proteins. In dot blot assay, hEGF was detected in both glycosylated and non-glycosylated fractions. Bioactivity assays revealed that both glycosylated and non-glycosylated fractions were bioactive as determined by cell viability assay. It was also observed that hEGF present in non-glycosylated fraction was relatively more bioactive than hEGF present in glycosylated fraction.     

Hypersensitive Reaction in Petunia

Intercellular fluid (IF) extracted from the apoplastic leaf spaces of Petunia contained up to 6 new soluble proteins, called d proteins (d₀ to d₅). Characterisation of the d proteins has shown that they can be divided into groups and show a genetic variability similar to the PR(b) proteins from Nicotiana. d protein patterns from Petunia interspecific hybrids clearly show that the determinant for the d proteins in sexually transmitted as is the case for b proteins in Nicotiana. d-protein induction in TMV-SM₁ infected Petunia leaves is discussed in relation to the resistance to infection shown by cultivars and hybrids of Petunia.     

Alpha-momorcharin enhances Nicotiana benthamiana resistance to tobacco mosaic virus infection through modulation of reactive oxygen species

Alpha-momorcharin (α-MMC), a member of the plant ribosomal inactivating proteins (RIPs) family, has been proven to exhibit important biological properties in animals, including antiviral, antimicrobial, and antitumour activities. However, the mechanism by which α-MMC increases plant resistance to viral infections remains unclear. To study the effect of α-MMC on plant viral defence and how α-MMC increases plant resistance to viruses, recombinant DNA and transgenic technologies were employed to investigate the role of α-MMC in Nicotiana benthamiana resistance to tobacco mosaic virus (TMV) infection. Treatment with α-MMC produced through DNA recombinant technology or overexpression of α-MMC mediated by transgenic technology alleviated TMV-induced oxidative damage and reduced the accumulation of reactive oxygen species (ROS) during TMV-green fluorescent protein infection of N. benthamiana. There was a significant decrease in TMV replication in the upper leaves following local α-MMC treatment and in α-MMC-overexpressing plants relative to control plants. These results suggest that application or overexpression of α-MMC in N. benthamiana increases resistance to TMV infection. Finally, our results showed that overexpression of α-MMC up-regulated the expression of ROS scavenging-related genes. α-MMC confers resistance to TMV infection by means of modulating ROS homeostasis through controlling the expression of antioxidant enzyme-encoding genes. Overall, our study revealed a new crosstalk mechanism between α-MMC and ROS during resistance to viral infection and provides a framework to understand the molecular mechanisms of α-MMC in plant defence against viral pathogens.     

Codon-modifications and an endoplasmic reticulum-targeting sequence additively enhance expression of an Aspergillus phytase gene in transgenic canola

Transgenic plants offer advantages for biomolecule production because plants can be grown on a large scale and the recombinant macromolecules can be easily harvested and extracted. We introduced an Aspergillus phytase gene into canola (Brassica napus) (line 9412 with low erucic acid and low glucosinolates) by Agrobacterium-mediated transformation. Phytase expression in transgenic plant was enhanced with a synthetic phytase gene according to the Brassica codon usage and an endoplasmic reticulum (ER) retention signal KDEL that confers an ER accumulation of the recombinant phytase. Secretion of the phytase to the extracellular fluid was also established by the use of the tobacco PR-S signal peptide. Phytase accumulation in mature seed accounted for 2.6% of the total soluble proteins. The enzyme can be glycosylated in the seeds of transgenic plants and retain a high stability during storage. These results suggest a commercial feasibility of producing a stable recombinant phytase in canola at a high level for animal feed supplement and for reducing phosphorus eutrophication problems.     

The Natural Occurrence of Turnip Mosaic Potyvirus in Allium ampeloprasum

An isolate of turnip mosaic potyvirus (TuMV) was obtained from Allium ampeloprasum grown in commercial greenhouses in Israel. Symptoms on infected plants include systemic chlorosis and yellow stripes, accompanied by growth reduction. Leaves were distorted, often showing necrotic flecking. The virus was readily transmitted mechanically, and in a non-persistent manner by aphids, among Allium, Chenopodium. Gomphrena and some Nicotiana spp. Purified preparations contained numerous filamentous particles similar to those observed in crude extracts of infected leaves. Particles from crude plant extracts had a normal length of 806 nm. Cells of infected plants contained cylindrical cytoplasmic inclusions with pinwheel, scrolls and laminated aggregates which indicated the presence of a potyvirus of Edwardson's subgroup III. and which resemble those of turnip mosaic virus (TuMV), The virus reacted strongly with antiserum to typical isolates of TuMV in immunoelectron microscopy and western blotting but not with antisera to several other potyviruses. Based on serological reactivity, electron microscopy, aphid transmission and cytopathology, the virus was identified as an isolate of TuMV.     

Secretion of biologically active glycoforms of bovine follicle stimulating hormone in plants.

We chose the follicle stimulating hormone (FSH), a pituitary heterodimeric glycoprotein hormone, as a model to assess the ability of the plant cell to express a recombinant protein that requires extensive N-glycosylation for subunit folding and assembly, intracellular trafficking, signal transduction and circulatory stability. A tobacco mosaic virus (TMV) based transient expression system was used to express a single-chain (sc) version of bovine FSH in the tobacco related species Nicotiana benthamiana. Preparations of periplasmic proteins from plants infected with recombinant viral RNA contained high levels of sc-bFSH, up to 3% of total soluble proteins. Consistently, in situ indirect immunofluorescence revealed that the plant cell secreted the mammalian secretory protein to the extracellular compartment (EC). By mass spectrometric analysis of immunoaffinity purified sc-bFSH derived from EC fractions, we found two species of the plant paucimannosidic glycan type, truncated forms of complex-type N-glycans. Stimulation of cAMP production in a CHO cell line expressing the porcine FSH receptor acknowledged the native-like structure of sc-bFSH and a sufficient extent of N-glycosylation required for signal transduction. Furthermore, in superovulatory treatments of mice, sc-bFSH displayed significant in vivo bioactivity, although much lower than that of pregnant mare serum gonadotropin. We conclude that plants may have a broad utility as hosts for the recombinant expression of proteins even where glycosylation is essential for function.     

Fusion with pep-1, a cell-penetrating peptide,enhances the transmembrane ability of human epidermal growth factor

Administration of macromolecule compositions in medicine and cosmetics always exhibited low bioavailability due to the limitation of transmembrane transport. Here, human epidermal growth factor (hEGF) was fused with glutathione S-transferase (GST) and Pep-1, the first commercial cell-penetrating peptide, in Escherichia coli. The fusion protein was firstly purified with the affinity chromatography, and then the GST tag was released by TEV protease. Final purification was achieved by the ion exchange chromatography. The biological activities and the transmembrane ability of the obtained products were determined using scratch wound-healing assay, MTT analysis, and immunofluorescence assay. The results showed that both rhEGF and Pep-1-fused hEGF were soluble expressed in E. coli. The fusion of Pep-1 could markedly increase the transmembrane ability of EGF, whereas it did not interfere with the growth-stimulating and migration-promoting functions of hEGF on fibroblasts. This research provided a novel strategy for the transmembrane transport of protein-derived cosmetics or drugs.     

Efficient transient expression of human GM-CSF protein in Nicotiana benthamiana using potato virus X vector

The human granulocyte macrophage colony-stimulating factor (GM-CSF) is a glycoprotein with important clinical applications for the treatment of neutropenia and aplastic anemia and reducing infections associated with bone marrow transplants. We evaluated the potential for using a potato virus X (PVX) viral vector system for efficient expression of the biologically functional GM-CSF protein in Nicotiana benthamiana leaves. The GM-CSF gene was cloned into PVX viral expression vector, driven with the CaMV 35S promoter. Gene transfer was accomplished by inoculating N. benthamiana leaves with the plasmid DNA of PVX vector containing the GM-CSF gene. The expression level of the recombinant GM-CSF protein was determined with ELISA and its size was confirmed by Western blot analysis. The results showed that: (1) leaf age significantly affects GM-CSF protein concentration with younger leaves accumulating 19.8 mg g⁻¹ soluble protein which is 2.6 times the concentration in older leaves, (2) recombinant protein accumulation within a given leaf declined slightly over time but was not significantly different between 7 and 11 days post-inoculation (dpi), and (3) the two leaves immediately above the inoculated leaves play an important role for GM-CSF accumulation in the younger leaves. Protein extracts of infected N. benthamiana leaves contained recombinant human GM-CSF protein in concentrations of up to 2% of total soluble protein, but only when the pair of leaves immediately above the inoculated leaves remained intact. The recombinant protein actively stimulated the growth of human TF-1 cells suggesting that the recombinant human GM-CSF expressed via PVX viral vector was biologically active.     

Exploring different strategies to express Dengue virus envelope protein in a plant system

Dengue virus envelope glycoprotein (E-protein) is the main protein associated with immunity induction. To produce a candidate for subunit vaccines and to provide an antigen for diagnostic kits, it was expressed in a novel plant system using deconstructed viral modules. A truncated version of the E-protein was designed to be expressed alone and co-expressed with Dengue virus structural proteins. As well, the critical domain III of E-protein was fused to hepatitis B core antigen (HBcore). The recombinant proteins were produced in Nicotiana benthamiana plants and were reactive with the anti-E antibody. The fusion was reactive with both anti-E and anti-HBcore antibodies.