scholarly journals Calreticulin co-expression supports high level production of a recombinant SARS-CoV-2 spike mimetic in Nicotiana benthamiana

2020 ◽  
Author(s):  
Emmanuel Margolin ◽  
Matthew Verbeek ◽  
Ann Meyers ◽  
Ros Chapman ◽  
Anna-Lise Williamson ◽  
...  
Keyword(s):  
Nicotiana Benthamiana ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Molecular Engineering ◽  
Plant Protease ◽  
Viral Glycoproteins ◽  
Human Proteins ◽  
Purified Antigen ◽  
High Level

AbstractAn effective prophylactic vaccine is urgently needed to protect against SARS-CoV-2 infection. The viral spike, which mediates entry into cells by interacting with the host angiotensin-converting enzyme 2, is the primary target of most vaccines in development. These vaccines aim to elicit protective immunity against the glycoprotein by use of inactivated virus, vector-mediated delivery of the antigen in vivo, or by direct immunization with the purified antigen following expression in a heterologous system. These approaches are mostly dependent on the growth of mammalian or insect cells, which requires a sophisticated infrastructure that is not generally available in developing countries due to the incumbent costs which are prohibitive. Plant-based subunit vaccine production has long been considered as a cheaper alternative, although low expression yields and differences along the secretory pathway to mammalian cells have posed a challenge to producing certain viral glycoproteins. Recent advances that have enabled many of these constraints to be addressed include expressing the requisite human proteins in plants to support the maturation of the protein of interest. In this study we investigated these approaches to support the production of a soluble and putatively trimeric SARS-CoV-2 spike mimetic in Nicotiana benthamiana via transient Agrobacterium-mediated expression. The co-expression of human calreticulin dramatically improved the accumulation of the viral spike, which was barely detectable in the absence of the co-expressed accessory protein. The viral antigen was efficiently processed even in the absence of co-expressed furin, suggesting that processing may have occurred at the secondary cleavage site and was mediated by an endogenous plant protease. In contrast, the spike was not efficiently processed when expressed in mammalian cells as a control, although the co-expression of furin improved processing considerably. This study demonstrates the feasibility of molecular engineering to improve the production of viral glycoproteins in plants, and supports plant-based production of SARS-CoV-2 spike-based vaccines and reagents for serological assays.

scholarly journals A Roadmap for the Molecular Farming of Viral Glycoprotein Vaccines: Engineering Glycosylation and Glycosylation-Directed Folding

2020 ◽  
Vol 11 ◽  
Author(s):  
Emmanuel Margolin ◽  
Max Crispin ◽  
Ann Meyers ◽  
Ros Chapman ◽  
Edward P. Rybicki
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Molecular Farming ◽  
Molecular Engineering ◽  
Early Years ◽  
In Planta ◽  
Post Translational Modification ◽  
Viral Glycoprotein ◽  
Viral Glycoproteins ◽  
Cellular Machinery

Immunization with recombinant glycoprotein-based vaccines is a promising approach to induce protective immunity against viruses. However, the complex biosynthetic maturation requirements of these glycoproteins typically necessitate their production in mammalian cells to support their folding and post-translational modification. Despite these clear advantages, the incumbent costs and infrastructure requirements with this approach can be prohibitive in developing countries, and the production scales and timelines may prove limiting when applying these production systems to the control of pandemic viral outbreaks. Plant molecular farming of viral glycoproteins has been suggested as a cheap and rapidly scalable alternative production system, with the potential to perform post-translational modifications that are comparable to mammalian cells. Consequently, plant-produced glycoprotein vaccines for seasonal and pandemic influenza have shown promise in clinical trials, and vaccine candidates against the newly emergent severe acute respiratory syndrome coronavirus-2 have entered into late stage preclinical and clinical testing. However, many other viral glycoproteins accumulate poorly in plants, and are not appropriately processed along the secretory pathway due to differences in the host cellular machinery. Furthermore, plant-derived glycoproteins often contain glycoforms that are antigenically distinct from those present on the native virus, and may also be under-glycosylated in some instances. Recent advances in the field have increased the complexity and yields of biologics that can be produced in plants, and have now enabled the expression of many viral glycoproteins which could not previously be produced in plant systems. In contrast to the empirical optimization that predominated during the early years of molecular farming, the next generation of plant-made products are being produced by developing rational, tailor-made approaches to support their production. This has involved the elimination of plant-specific glycoforms and the introduction into plants of elements of the biosynthetic machinery from different expression hosts. These approaches have resulted in the production of mammalian N-linked glycans and the formation of O-glycan moieties in planta. More recently, plant molecular engineering approaches have also been applied to improve the glycan occupancy of proteins which are not appropriately glycosylated, and to support the folding and processing of viral glycoproteins where the cellular machinery differs from the usual expression host of the protein. Here we highlight recent achievements and remaining challenges in glycoengineering and the engineering of glycosylation-directed folding pathways in plants, and discuss how these can be applied to produce recombinant viral glycoproteins vaccines.

scholarly journals The multiple facets of the Golgi reassembly stacking proteins

2011 ◽  
Vol 433 (3) ◽  
pp. 423-433 ◽  
Author(s):  
Fabian P. Vinke ◽  
Adam G. Grieve ◽  
Catherine Rabouille
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Mitotic Checkpoint ◽  
Biochemical Properties ◽  
C Terminus ◽  
Unconventional Secretion ◽  
Golgi Ribbon

The mammalian GRASPs (Golgi reassembly stacking proteins) GRASP65 and GRASP55 were first discovered more than a decade ago as factors involved in the stacking of Golgi cisternae. Since then, orthologues have been identified in many different organisms and GRASPs have been assigned new roles that may seem disconnected. In vitro, GRASPs have been shown to have the biochemical properties of Golgi stacking factors, but the jury is still out as to whether they act as such in vivo. In mammalian cells, GRASP65 and GRASP55 are required for formation of the Golgi ribbon, a structure which is fragmented in mitosis owing to the phosphorylation of a number of serine and threonine residues situated in its C-terminus. Golgi ribbon unlinking is in turn shown to be part of a mitotic checkpoint. GRASP65 also seems to be the key target of signalling events leading to re-orientation of the Golgi during cell migration and its breakdown during apoptosis. Interestingly, the Golgi ribbon is not a feature of lower eukaryotes, yet a GRASP homologue is present in the genome of Encephalitozoon cuniculi, suggesting they have other roles. GRASPs have no identified function in bulk anterograde protein transport along the secretory pathway, but some cargo-specific trafficking roles for GRASPs have been discovered. Furthermore, GRASP orthologues have recently been shown to mediate the unconventional secretion of the cytoplasmic proteins AcbA/Acb1, in both Dictyostelium discoideum and yeast, and the Golgi bypass of a number of transmembrane proteins during Drosophila development. In the present paper, we review the multiple roles of GRASPs.

Molecular engineering of Surfaces Using Self-Assembled Monolayers

2005 ◽  
Vol 88 (1) ◽  
pp. 17-48 ◽  
Author(s):  
George M. Whitesides ◽  
Jennah K. Kriebel ◽  
J. Christopher Love
Keyword(s):  
Surface Science ◽  
Self Assembly ◽  
Mammalian Cells ◽  
Molecular Level ◽  
Level Structure ◽  
Molecular Engineering ◽  
Gold Silver ◽  
Wide Range ◽  
Macroscopic Properties ◽  
High Level

The self-assembly of molecules into structurally organized monolayers (SAMs) uses the flexibility of organic chemistry and coordination chemistry to generate well-defined, synthetic surfaces with known molecular and macroscopic properties. The process of designing monolayers with a specified structure gives a high level of control over the molecular-level composition in the direction perpendicular to a surface; soft lithographic technique gives useful (if lower) resolution in the plane of the surface. Alkanethiolates adsorbed on gold, silver, mercury, palladium and platinum are currently the best-defined systems of SAMs. They provide substrates for a number of applications-from studies of wetting and electron transport to patterns for growing mammalian cells. SAMs have made organic surfaces a central part of surface science. Understanding the principles by which they form, and connecting molecular-level structure with macroscopic properties, opens a wide range of areas to study and exploitation.

scholarly journals Identification of Novel Interaction Partners of AIF Protein on the Outer Mitochondrial Membrane

Acta Naturae ◽  
2018 ◽  
Vol 10 (4) ◽  
pp. 100-109 ◽  
Author(s):  
N. P. Fadeeva ◽  
N. V. Antipova ◽  
V. O. Shender ◽  
K. S. Anufrieva ◽  
G. A. Stepanov ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mitochondrial Membrane ◽  
Bioinformatic Analysis ◽  
Amino Acid Sequences ◽  
Outer Mitochondrial Membrane ◽  
Apoptotic Pathway ◽  
Survival Prognosis ◽  
High Level

In response to the wide variety of external and internal signals, mammalian cells undergo apoptosis, programmed cell death. Dysregulation of apoptosis is involved in multiple human diseases, including cancer, autoimmunity, and ischemic injuries. Two types of apoptosis have been described: the caspase-dependent one, leading to digestion of cellular proteins, and caspase-independent apoptosis, resulting in DNA fragmentation. The latter type of apoptosis is executed by AIF protein and is believed to have appeared first during evolution. The key step in the caspase-independent apoptosis program is the dissociation of AIF from the outer mitochondrial membrane (OMM). However, the molecular mechanism of interaction between AIF and OMM remains poorly understood. In this study, we demonstrated that AIF can bind to OMM via mortalin protein. We confirmed interaction between AIF and mortalin both in vitro and in vivo and mapped the amino acid sequences that are important for the binding of these proteins. Next, we showed that apoptosis induction by chemotherapy leads to downregulation of AIF-mortalin interaction and dissociation of AIF from the OMM. Finally, a bioinformatic analysis demonstrated that a high level of mortalin expression correlates with a worse survival prognosis for glioma patients. Altogether, our data revealed that mortalin plays an important role in the regulation of the caspase-independent apoptotic pathway and allowed us to speculate that inhibition of AIF-mortalin interaction may induce a dissociation of AIF from the OMM and subsequent apoptosis of cancer cells.

scholarly journals Mammalian Y RNAs are modified at discrete guanosine residues with N-glycans

2019 ◽  
Author(s):  
Ryan A. Flynn ◽  
Benjamin A. H. Smith ◽  
Alex G. Johnson ◽  
Kayvon Pedram ◽  
Benson M. George ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Cultured Cells ◽  
Mammalian Species ◽  
Cell Types ◽  
Intramolecular Interactions ◽  
Small Noncoding Rnas ◽  
Select Group ◽  
Domains Of Life

ABSTRACTGlycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA, another multifaceted biopolymer, is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammalian cells use RNA as a third scaffold for glycosylation in the secretory pathway. Using a battery of chemical and biochemical approaches, we find that a select group of small noncoding RNAs including Y RNAs are modified with complex, sialylated N-glycans (glycoRNAs). These glycoRNA are present in multiple cell types and mammalian species, both in cultured cells andin vivo. Finally, we find that RNA glycosylation depends on the canonical N-glycan biosynthetic machinery within the ER/Golgi luminal spaces. Collectively, these findings suggest the existence of a ubiquitous interface of RNA biology and glycobiology suggesting an expanded role for glycosylation beyond canonical lipid and protein scaffolds.

scholarly journals Site-Specific Glycosylation of Recombinant Viral Glycoproteins Produced in Nicotiana benthamiana

2021 ◽  
Vol 12 ◽  
Author(s):  
Emmanuel Margolin ◽  
Joel D. Allen ◽  
Matthew Verbeek ◽  
Michiel van Diepen ◽  
Phindile Ximba ◽  
...  
Keyword(s):  
Mammalian Cell ◽  
Nicotiana Benthamiana ◽  
Mammalian Cells ◽  
Large Scale ◽  
Molecular Farming ◽  
Marburg Virus ◽  
Viral Envelope ◽  
Scale Production ◽  
Site Specific ◽  
Viral Glycoproteins

There is an urgent need to establish large scale biopharmaceutical manufacturing capacity in Africa where the infrastructure for biologics production is severely limited. Molecular farming, whereby pharmaceuticals are produced in plants, offers a cheaper alternative to mainstream expression platforms, and is amenable to rapid large-scale production. However, there are several differences along the plant protein secretory pathway compared to mammalian systems, which constrain the production of complex pharmaceuticals. Viral envelope glycoproteins are important targets for immunization, yet in some cases they accumulate poorly in plants and may not be properly processed. Whilst the co-expression of human chaperones and furin proteases has shown promise, it is presently unclear how plant-specific differences in glycosylation impact the production of these proteins. In many cases it may be necessary to reproduce features of their native glycosylation to produce immunologically relevant vaccines, given that glycosylation is central to the folding and immunogenicity of these antigens. Building on previous work, we transiently expressed model glycoproteins from HIV and Marburg virus in Nicotiana benthamiana and mammalian cells. The proteins were purified and their site-specific glycosylation was determined by mass-spectrometry. Both glycoproteins yielded increased amounts of protein aggregates when produced in plants compared to the equivalent mammalian cell-derived proteins. The glycosylation profiles of the plant-produced glycoproteins were distinct from the mammalian cell produced proteins: they displayed lower levels of glycan occupancy, reduced complex glycans and large amounts of paucimannosidic structures. The elucidation of the site-specific glycosylation of viral glycoproteins produced in N. benthamiana is an important step toward producing heterologous viral glycoproteins in plants with authentic human-like glycosylation.

scholarly journals A human protein selected for interference with Ras function interacts directly with Ras and competes with Raf1.

1995 ◽  
Vol 15 (3) ◽  
pp. 1318-1323 ◽  
Author(s):  
L Han ◽  
J Colicelli
Keyword(s):  
Mammalian Cells ◽  
Signal Transduction Pathway ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Yeast Saccharomyces Cerevisiae ◽  
Activating Mutations ◽  
Human Proteins ◽  
Ras Signal Transduction

The overexpression of some human proteins can cause interference with the Ras signal transduction pathway in the yeast Saccharomyces cerevisiae. The functional block is located at the level of the effector itself, since these proteins do not suppress activating mutations further downstream in the same pathway. We now demonstrate, with in vivo and in vitro experiments, that the protein encoded by one human cDNA (clone 99) can interact directly with yeast Ras2p and with human H-Ras protein, and we have named this gene rin1 (Ras interaction/interference). The interaction between Ras and Rin1 is enhanced when Ras is bound to GTP. Rin1 is not able to interact with either an effector mutant or a dominant negative mutant of H-Ras. Thus, Rin1 displays a human H-Ras interaction profile that is the same as that seen for Raf1 and yeast adenylyl cyclase, two known effectors of Ras. Moreover, Raf1 directly competes with Rin1 for binding to H-Ras in vitro. Unlike Raf1, however, the Rin1 protein resides primarily at the plasma membrane, where H-Ras is localized. These data are consistent with Rin1 functioning in mammalian cells as an effector or regulator of H-Ras.

scholarly journals Protection of bone marrow transplant recipients from lethal doses of methotrexate by the generation of methotrexate-resistant bone marrow.

1987 ◽  
Vol 166 (1) ◽  
pp. 210-218 ◽  
Author(s):  
D A Williams ◽  
K Hsieh ◽  
A DeSilva ◽  
R C Mulligan
Keyword(s):  
Bone Marrow ◽  
Mammalian Cells ◽  
Bone Marrow Cells ◽  
Marrow Transplant ◽  
Murine Bone Marrow ◽  
Vitro Analysis ◽  
High Level ◽  
Lethal Doses

To develop a highly efficient means for generating methotrexate resistant (MTXr) hematopoietic cells in vivo, a recombinant retroviral genome was constructed that encodes a MTXr dihydrofolate reductase (DHFRr). Cell lines producing high titers of virus capable of transmitting the DHFR gene were generated and used to infect mammalian cells in vitro. Analysis of infected fibroblasts indicated that the DHFRr gene was transmitted intact and conferred a high level of MTXr upon cells. Based on these findings, DHFRr-containing virus was used to infect murine bone marrow cells in vitro. Following infection, the transduced cells were introduced into lethally irradiated recipients via bone marrow transplantation techniques. The presence of the proviral sequences in cells of the spleen and bone marrow of engrafted recipients was associated with significantly increased survival of mice treated with otherwise lethal doses of MTX.

scholarly journals Expression of genes coding for animal virus glycoproteins in heterologous systems.

1999 ◽  
Vol 46 (2) ◽  
pp. 325-339 ◽  
Author(s):  
K Bieńkowska-Szewczyk ◽  
B Szewczyk
Keyword(s):  
Mammalian Cells ◽  
Insect Cells ◽  
Host Cells ◽  
Animal Virus ◽  
Expression Of Genes ◽  
Viral Glycoproteins ◽  
Foreign Genes ◽  
Nuclear Polyhedrosis ◽  
High Level ◽  
Expression In Mammalian Cells

The outermost layers of animal viruses are usually composed of glycoproteins. They are responsible not only for the entrance of viruses into, and release from host cells but also for the initial interaction of a viral particle with immunological defense of the host. It is therefore not surprising that many laboratories devote a lot of effort to study viral glycoproteins at the molecular level. Very often such studies are possible only after the introduction of a glycoprotein gene into a heterologous system. Expression of glycoprotein genes is usually obtained in mammalian or insect cells. Expression in mammalian cells yields viral glycoproteins with glycan chains indistinguishable from the original counterparts in virion particles but the level of synthesis of glycoproteins is very low. Vaccinia virus is the most common vector for expression in mammalian cells. It is easy to grow, the introduction of foreign genes is relatively simple and, due to the size of the vaccinia genome, it can accept large pieces of foreign DNA. Glycosylation in insect cells is not as complex as in mammalian cells and usually glycoproteins produced in insect cells are of slightly lower molecular mass than those produced in mammalian cells. The most common vector for expression of glycoproteins in insect cells is a baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). The great advantage of this system is a very high level of expression of foreign genes.

scholarly journals Durable multitransgene expression in vivo using systemic, nonviral DNA delivery

2019 ◽  
Vol 5 (11) ◽  
pp. eaax0217 ◽  
Author(s):  
Chakkrapong Handumrongkul ◽  
Alice L. Ye ◽  
Stephen A. Chmura ◽  
Liliana Soroceanu ◽  
Marissa Mack ◽  
...  
Keyword(s):  
Immune Responses ◽  
Therapeutic Potential ◽  
Expression System ◽  
System A ◽  
Human Proteins ◽  
Delivery Platform ◽  
Immunocompetent Mice ◽  
High Level ◽  
Deficiency Diseases

Recombinant adeno-associated virus (AAV) vectors are transforming therapies for rare human monogenic deficiency diseases. However, adaptive immune responses to AAV and its limited DNA insert capacity, restrict their therapeutic potential. HEDGES (high-level extended duration gene expression system), a nonviral DNA- and liposome-based gene delivery platform, overcomes these limitations in immunocompetent mice. Specifically, one systemic HEDGES injection durably produces therapeutic levels of transgene-encoded human proteins, including FDA–approved cytokines and monoclonal antibodies, without detectable integration into genomic DNA. HEDGES also controls protein production duration from <3 weeks to >1.5 years, does not induce anti-vector immune responses, is reexpressed for prolonged periods following reinjection, and produces only transient minimal toxicity. HEDGES can produce extended therapeutic levels of multiple transgene-encoded therapeutic human proteins from DNA inserts >1.5-fold larger than AAV-based therapeutics, thus creating combinatorial interventions to effectively treat common polygenic diseases driven by multigenic abnormalities.

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