Production of Recombinant Proteins in Transgenic Tobacco Plants.

Nicotiana tabacum (the tobacco plant ) has numerous advantages for molecular farming, including rapid growth, large biomass and the possibility of both cross- and self-fertilization. In addition, genetic transformation and tissue culture protocols for regeneration of transgenic plants are well-established. Here, we describe the production of transgenic tobacco using Agrobacterium tumefaciens and the analysis of recombinant proteins, either in crude plant extracts or after purification, by enzyme-linked immunosorbent assays, sodium dodecyl sulfate polyacrylamide gel electrophoresis with western blotting and surface plasmon resonance.

Molecular Pharming for low and middle income countries.

Interest in applications and benefits that Molecular Pharming might offer to Low and Middle Income Countries has always been a potent driver for the research discipline, and a major reason why many scientists entered the field. Although enthusiasm remains high, the reality is that such a game-changing innovation would always take longer than traditional uptake of new technology in developed countries, and be complicated by external factors beyond technical feasibility. Excitingly, signs of increasing interest by LMICS in Molecular Pharming are now emerging. Here, three case studies from Thailand, South Africa and Brazil are used to identify some of the key issues when a new investment into Molecular Pharming manufacturing capacity is under consideration. At present, academic research is not necessarily addressing these issues. Only by understanding the concerns, can members of the academic community contribute to helping the development of Molecular Pharming for LMICs by focusing their research efforts appropriately.

In vitro and in vivo evaluation of a single chain antibody fragment generated in planta with potent rabies neutralisation activity.

Rabies causes more than 60,000 human deaths annually in areas where the virus is endemic. Importantly, rabies is one of the few pathogens for which there is no treatment following the onset of clinical disease with the outcome of infection being death in almost 100% of cases. Whilst vaccination, and the combination of vaccine and rabies immunoglobulin treatment for post-exposure administration are available, no tools have been identified that can reduce or prevent rabies virus replication once clinical disease has initiated. The search for effective antiviral molecules to treat those that have already developed clinical disease associated with rabies virus infection is considered one of the most important goals in rabies research. The current study assesses a single chain antibody molecule (ScFv) based on a monoclonal antibody that potently neutralises rabies in vitro as a potential therapeutic candidate. The recombinant ScFv was generated in Nicotiana benthamiana by transient expression, and was chemically conjugated (ScFv/RVG) to a 29 amino acid peptide, specific for nicotinic acetylcholine receptor (nAchR) binding in the CNS. This conjugated molecule was able to bind nAchR in vitro and enter neuronal cells more efficiently than ScFv. The ability of the ScFv/RVG to neutralise virus in vivo was assessed using a staggered administration where the molecule was inoculated either four hours before, two days after or four days after infection. The ScFv/RVG conjugate was evaluated in direct comparison with HRIG and a potential antiviral molecule, Favipiravir (also known as T-705) to indicate whether there was greater bioavailability of the ScFv in the brains of treated mice. The study indicated that the approach taken with the ScFv/RVG conjugate may have utility in the design and implementation of novel tools targetting rabies virus infection in the brain.

Recombinant biologic products versus nutraceuticals from plants - a regulatory choice?

Biotechnology has transformed the potential for plants to be a manufacturing source of pharmaceutical compounds. Now, with transgenic and transient expression techniques, virtually any biologic, including vaccines and therapeutics, could be manufactured in plants. However, uncertainty over the regulatory path for such new pharmaceuticals has been a deterrent. Consideration has been given to using alternative regulatory paths, including those for nutraceuticals or cosmetic agents. This review will consider these possibilities, and discuss the difficulties in establishing regulatory guidelines for new pharmaceutical manufacturing technologies.

Rhizosecretion improves the production of Cyanovirin-N in Nicotiana tabacum through simplified downstream processing.

Rhizosecretion has many advantages for the production of recombinant pharmaceuticals, notably facile downstream processing from hydroponic medium. The aim of this study was to increase yields of the HIV microbicide candidate, Cyanovirin-N (CV-N), obtained using this production platform and to develop a simplified methodology for its downstream processing from hydroponic medium. Placing hydroponic cultures on an orbital shaker more than doubled the concentration of CV-N in the hydroponic medium compared to plants which remained stationary, reaching a maximum of approximately 20µg/ml in one week, which is more than 3 times higher than previously reported yields. The protein composition of the hydroponic medium, the rhizosecretome, was characterised in plants cultured with or without the plant growth regulator alpha-napthaleneacetic acid by LC-ESI-MS/MS, and CV-N was the most abundant protein. The issue of large volumes in the rhizosecretion system was addressed by using ion exchange chromatography to concentrate CV-N and partially remove impurities. The semi-purified CV-N was demonstrated to bind to HIV gp120 in an ELISA and to neutralise HIVBa-L with an IC50 of 6nM in a cell-based assay. Rhizosecretion is therefore a practicable and inexpensive method for the production of functional CV-N.

High-yield production of a human monoclonal IgG by rhizosecretion in hydroponic tobacco cultures.

Rhizosecretion of recombinant pharmaceuticals from in vitro hydroponic transgenic plant cultures is a simple, low cost, reproducible and controllable production method. Here, we demonstrate the application and adaptation of this manufacturing platform to a human antivitronectin IgG1 monoclonal antibody (mAb) called M12. The rationale for specific growth medium additives was established by phenotypic analysis of root structure and by LC-ESI-MS/MS profiling of the total protein content profile of the hydroponic medium. Through a combination of optimization approaches, mAb yields in hydroponic medium reached 46 µg/mL in 1 week, the highest figure reported for a recombinant mAb in a plant secretion-based system to date. The rhizosecretome was determined to contain 104 proteins, with the mAb heavy and light chains the most abundant. This enabled evaluation of a simple, scalable extraction and purification protocol and demonstration that only minimal processing was necessary prior to protein A affinity chromatography. MALDI-TOF MS revealed that purified mAb contained predominantly complex-type plant N-glycans, in three major glycoforms. The binding of M12 purified from hydroponic medium to vitronectin was comparable to its Chinese hamster ovary (CHO)-derived counterpart. This study demonstrates that in vitro hydroponic cultivation coupled with recombinant protein rhizosecretion can be a practical, low-cost production platform for monoclonal antibodies.

Transformation of Althaea officinalis L. by Agrobacterium rhizogenes for the production of transgenic roots expressing the anti-HIV microbicide cyanovirin-N.

The marshmallow plant (Althaea officinalis L.) has been used for centuries in medicine and other applications. Valuable secondary metabolites have previously been identified in Agrobacterium rhizogenes-generated transgenic 'hairy' roots in this species. In the present study, transgenic roots were produced in A. officinalis using A. rhizogenes. In addition to wild-type lines, roots expressing the anti-human immunodeficiency virus microbicide candidate, cyanovirin-N (CV-N), were generated. Wild-type and CV-N root lines were transferred to liquid culture and increased in mass by 49 and 19 % respectively over a 7 day culture period. In the latter, the concentration of CV-N present in the root tissue was 2.4 µg/g fresh weight, with an average secretion rate into the growth medium of 0.02 µg/ml/24 h. A. officinalis transgenic roots may therefore in the future be used not only as a source of therapeutic secondary metabolites, but also as an expression system for the production of recombinant pharmaceuticals.

Structure/function analysis of Neisseria meningitidis PilW, a conserved protein that plays multiple roles in type IV pilus biology.

Type IV pili (Tfp) are widespread filamentous bacterial organelles that mediate multiple functions and play a key role in pathogenesis in several important human pathogens, including Neisseria meningitidis. Tfp biology remains poorly understood at a molecular level because the roles of the numerous proteins that are involved remain mostly obscure. Guided by the high-resolution crystal structure we recently reported for N. meningitidis PilW, a widely conserved protein essential for Tfp biogenesis, we have performed a structure/function analysis by targeting a series of key residues through site-directed mutagenesis and analyzing the corresponding variants using an array of phenotypic assays. Here we show that PilW's involvement in the functionality of Tfp can be genetically uncoupled from its concurrent role in the assembly/stabilization of the secretin channels through which Tfp emerge on the bacterial surface. These findings suggest that PilW is a multifunctional protein.

Preparation of monoclonal antibodies against the spindle checkpoint kinase Bub1.

The Bub1 kinase is a critical component of the spindle checkpoint involved in monitoring the separation of sister chromatids at mitosis. The viral oncoprotein Simian virus 40 large T antigen (LT) can bind and perturb the spindle checkpoint function of Bub1. We have developed three highly specific monoclonal antibodies against the Bub1 protein and have demonstrated that they can all detect Bub1 via Western blotting and immunofluorescence, in addition to their ability to immunoprecipitate Bub1.

Discovery of an MIT-like atracotoxin family: spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins.

This project identified a novel family of six 66-68 residue peptides from the venom of two Australian funnel-web spiders, Hadronyche sp. 20 and H. infensa: Orchid Beach (Hexathelidae: Atracinae), that appear to undergo N- and/or C-terminal post-translational modifications and conform to an ancestral protein fold. These peptides all show significant amino acid sequence homology to atracotoxin-Hvf17 (ACTX-Hvf17), a non-toxic peptide isolated from the venom of H. versuta, and a variety of AVIT family proteins including mamba intestinal toxin 1 (MIT1) and its mammalian and piscine orthologs prokineticin 1 (PK1) and prokineticin 2 (PK2). These AVIT family proteins target prokineticin receptors involved in the sensitization of nociceptors and gastrointestinal smooth muscle activation. Given their sequence homology to MIT1, we have named these spider venom peptides the MIT-like atracotoxin (ACTX) family. Using isolated rat stomach fundus or guinea-pig ileum organ bath preparations we have shown that the prototypical ACTX-Hvf17, at concentrations up to 1muM, did not stimulate smooth muscle contractility, nor did it inhibit contractions induced by human PK1 (hPK1). The peptide also lacked activity on other isolated smooth muscle preparations including rat aorta. Furthermore, a FLIPR Ca2+ flux assay using HEK293 cells expressing prokineticin receptors showed that ACTX-Hvf17 fails to activate or block hPK1 or hPK2 receptors. Therefore, while the MIT-like ACTX family appears to adopt the ancestral disulfide-directed beta-hairpin protein fold of MIT1, a motif believed to be shared by other AVIT family peptides, variations in the amino acid sequence and surface charge result in a loss of activity on prokineticin receptors.

The MinD membrane targeting sequence is a transplantable lipid-binding helix.

MinD is a ubiquitous ATPase that plays a crucial role in selection of the division site in eubacteria, chloroplasts, and probably also Archaea. It was recently demonstrated that membrane localization of MinD is mediated by an 8-12-residue C-terminal motif termed the membrane targeting sequence or MTS. In this study we show that the MinD MTS is a transplantable lipid-binding motif that can effectively target heterologous proteins to the cell membrane. We demonstrate that eubacterial MTSs interact directly with lipid bilayers as an amphipathic helix, with a distinct preference for anionic phospholipids. Moreover, we provide evidence that the phospholipid preference of each MTS, as well as its affinity for biological membranes, has been evolutionarily "tuned" to its specific role in different bacteria. We propose a model to describe how the MTS is coupled to ATP binding to regulate the reversible membrane association of Escherichia coli MinD during its pole-to-pole oscillation cycle.

Membrane localization of MinD is mediated by a C-terminal motif that is conserved across eubacteria, archaea, and chloroplasts.

MinD is a widely conserved ATPase that has been demonstrated to play a pivotal role in selection of the division site in eubacteria and chloroplasts. It is a member of the large ParA superfamily of ATPases that are characterized by a deviant Walker-type ATP-binding motif. MinD localizes to the cytoplasmic face of the inner membrane in Escherichia coli, and its association with the inner membrane is a prerequisite for membrane recruitment of the septation inhibitor MinC. However, the mechanism by which MinD associates with the membrane has proved enigmatic; it seems to lack a transmembrane domain and the amino acid sequence is devoid of hydrophobic tracts that might predispose the protein to interaction with lipids. In this study, we show that the extreme C-terminal region of MinD contains a highly conserved 8- to 12-residue sequence motif that is essential for membrane localization of the protein. We provide evidence that this motif forms an amphipathic helix that most likely mediates a direct interaction between MinD and membrane phospholipids. A model is proposed whereby the membrane-targeting motif mediates the rapid cycles of membrane attachment-release-reattachment that are presumed to occur during pole-to-pole oscillation of MinD in E. coli.