Gene Expression
Plant Viral Vectors
Plant Viral Vectors for Protein Expression
from Yuri Y. Gleba and Anatoli Giritch writing in Recent Advances in Plant Virology
Plant-virus-driven transient expression of heterologous proteins is the basis of several mature manufacturing processes that are currently being used for the production of multiple proteins including vaccine antigens and antibodies. Viral vectors have also become useful tools for research. In recent years, advances have been made both in the development of first-generation vectors (those that employ the 'full virus' strategy) as well as second-generation vectors designed using the 'deconstructed virus' approach. This second strategy relies on Agrobacterium as a vector to deliver DNA copies of one or more viral RNA replicons. Among the most often used viral backbones are those of Tobacco mosaic virus, Potato virus X, and Cowpea mosaic virus. Prototypes of industrial processes that provide for high-yield, rapid scale-up, and fast manufacturing have been recently developed using viral vectors, with several manufacturing facilities compliant with good manufacturing practices (GMP) in place, and a number of pharmaceutical proteins currently in pre-clinical and clinical trials.
Further reading: Recent Advances in Plant Virology | Virology Publications
from Yuri Y. Gleba and Anatoli Giritch writing in Recent Advances in Plant Virology
Plant-virus-driven transient expression of heterologous proteins is the basis of several mature manufacturing processes that are currently being used for the production of multiple proteins including vaccine antigens and antibodies. Viral vectors have also become useful tools for research. In recent years, advances have been made both in the development of first-generation vectors (those that employ the 'full virus' strategy) as well as second-generation vectors designed using the 'deconstructed virus' approach. This second strategy relies on Agrobacterium as a vector to deliver DNA copies of one or more viral RNA replicons. Among the most often used viral backbones are those of Tobacco mosaic virus, Potato virus X, and Cowpea mosaic virus. Prototypes of industrial processes that provide for high-yield, rapid scale-up, and fast manufacturing have been recently developed using viral vectors, with several manufacturing facilities compliant with good manufacturing practices (GMP) in place, and a number of pharmaceutical proteins currently in pre-clinical and clinical trials.
Further reading: Recent Advances in Plant Virology | Virology Publications
Thiol-based sensory factors
from Haike Antelmann and Peter Zuber in Sensory Mechanisms in Bacteria: Molecular Aspects of Signal Recognition
Bacteria regularly encounter Reactive Oxygen, Nitrogen and Electrophilic Species (ROS, RNS, RES) that are generated inside the cells by incomplete reduction of molecular oxygen, imbalanced metabolic processes or applied externally by toxic or antimicrobial compounds. The response to such reactive agents is mediated by redox-sensitive transcription factors that exploit the unique chemistry of cysteine thiol groups. Redox-sensitive regulatory proteins bear cysteine residues that can undergo post-translational modification, leading to either activation or inactivation of the transcription factors. This in turn results in responses that are aimed to detoxify the reactive species or alleviate the damage they cause. Different thiol-modifications are implicated in redox-sensing depending on the number of redox-active Cys residues and their reactivity, the oxidant to which they react, and the prevailing in vitro or in vivo conditions. Redox-sensitive proteins with more than one reactive Cys residue undergo in most cases reversible inter- and/or intramolecular disulfide linkages, which serve as sensing mechanisms for OxyR, the 2-Cys OhrR family, MexR, OspR, Spx, CprK and CrtJ. In contrast to these classical thiol-disulfide-switches, transcription factors with one redox-active Cys residue are reversibly regulated via initial sulphenic acid formation, S-thiolation with low molecular weight (LMW) thiols and sulfenamide formation with the backbone amide as shown for OxyR, the 1-Cys OhrR ortholog, MgrA and SarZ. However, the thiol group of the 1-Cys OhrR protein can also be irreversibly modified by overoxidation to sulfinic and sulfonic acids in response to strong oxidants. RES such as quinones were shown to modify the YodB repressor irreversibly by thiol-(S)-alkylation. In addition to redox-sensing transcription factors, LMW thiols and the thioredoxin/thioredoxin reductase system maintain the thiol-redox-balance of the cell upon exposure to reactive species. Here we review (1) enzymatic redox control mechanisms by thiol-disulfide reductases and (2) the current knowledge of bacterial redox-sensitive transcription factors that function without metal cofactors, including OxyR, OhrR, MexR, OspR, MgrA, SarZ, YodB, Spx, CprK and PspR/CrtJ. Each of these transcription factors senses unique signals including ROS, RNS, RES, antibiotic and haloorganic compounds, or the cellular oxygen level and light that are transduced via diverse redox-sensing mechanisms involving different reversible and irreversible thiol-modifications.
Further reading: Sensory Mechanisms in Bacteria: Molecular Aspects of Signal Recognition
Bacteria regularly encounter Reactive Oxygen, Nitrogen and Electrophilic Species (ROS, RNS, RES) that are generated inside the cells by incomplete reduction of molecular oxygen, imbalanced metabolic processes or applied externally by toxic or antimicrobial compounds. The response to such reactive agents is mediated by redox-sensitive transcription factors that exploit the unique chemistry of cysteine thiol groups. Redox-sensitive regulatory proteins bear cysteine residues that can undergo post-translational modification, leading to either activation or inactivation of the transcription factors. This in turn results in responses that are aimed to detoxify the reactive species or alleviate the damage they cause. Different thiol-modifications are implicated in redox-sensing depending on the number of redox-active Cys residues and their reactivity, the oxidant to which they react, and the prevailing in vitro or in vivo conditions. Redox-sensitive proteins with more than one reactive Cys residue undergo in most cases reversible inter- and/or intramolecular disulfide linkages, which serve as sensing mechanisms for OxyR, the 2-Cys OhrR family, MexR, OspR, Spx, CprK and CrtJ. In contrast to these classical thiol-disulfide-switches, transcription factors with one redox-active Cys residue are reversibly regulated via initial sulphenic acid formation, S-thiolation with low molecular weight (LMW) thiols and sulfenamide formation with the backbone amide as shown for OxyR, the 1-Cys OhrR ortholog, MgrA and SarZ. However, the thiol group of the 1-Cys OhrR protein can also be irreversibly modified by overoxidation to sulfinic and sulfonic acids in response to strong oxidants. RES such as quinones were shown to modify the YodB repressor irreversibly by thiol-(S)-alkylation. In addition to redox-sensing transcription factors, LMW thiols and the thioredoxin/thioredoxin reductase system maintain the thiol-redox-balance of the cell upon exposure to reactive species. Here we review (1) enzymatic redox control mechanisms by thiol-disulfide reductases and (2) the current knowledge of bacterial redox-sensitive transcription factors that function without metal cofactors, including OxyR, OhrR, MexR, OspR, MgrA, SarZ, YodB, Spx, CprK and PspR/CrtJ. Each of these transcription factors senses unique signals including ROS, RNS, RES, antibiotic and haloorganic compounds, or the cellular oxygen level and light that are transduced via diverse redox-sensing mechanisms involving different reversible and irreversible thiol-modifications.
Further reading: Sensory Mechanisms in Bacteria: Molecular Aspects of Signal Recognition
Sensory Mechanisms in Bacteria
from Sensory Mechanisms in Bacteria: Molecular Aspects of Signal Recognition
Bacteria have evolved extraordinary abilities to detect physical and chemical signals, both within their own cells and in the extracellular environment. The interaction of a signal with its receptor (usually a protein or RNA molecule) triggers a series of events that lead to reprogramming of cellular physiology, typically as a consequence of altered patterns of gene expression. In this way, the bacterial cell is able to mount appropriate and effective responses to changing physical and/or chemical environments. The versatility with which many bacteria adapt to environmental change underlies many important aspects of microbiology. For example, pathogens encounter multiple environments as they invade a host from the outside, and then progress through different sites within host tissues. There is growing evidence that pathogenic bacteria make use of physical and chemical cues to signal their presence in a suitable host, and need to adapt to the host environment in order to mount a successful infection. On the other hand, it should not be assumed that all signals to which bacteria must respond originate in the extracellular environment. For many species, even the cosseted life in a laboratory shake flask is 'stressful', in the sense that there is often a need to avoid or reverse the effects of harmful intermediates or by-products of metabolism. For example, all organisms that use dioxygen as a terminal electron acceptor have to deal with the reactive oxygen species that arise as adventitious by-products of aerobic metabolism. In bacteria, multiple protein receptors for oxygen radicals have been described, which control the expression of genes encoding enzymes that detoxify oxygen radicals or repair the damage that they cause.
Further reading: Sensory Mechanisms in Bacteria: Molecular Aspects of Signal Recognition
Bacteria have evolved extraordinary abilities to detect physical and chemical signals, both within their own cells and in the extracellular environment. The interaction of a signal with its receptor (usually a protein or RNA molecule) triggers a series of events that lead to reprogramming of cellular physiology, typically as a consequence of altered patterns of gene expression. In this way, the bacterial cell is able to mount appropriate and effective responses to changing physical and/or chemical environments. The versatility with which many bacteria adapt to environmental change underlies many important aspects of microbiology. For example, pathogens encounter multiple environments as they invade a host from the outside, and then progress through different sites within host tissues. There is growing evidence that pathogenic bacteria make use of physical and chemical cues to signal their presence in a suitable host, and need to adapt to the host environment in order to mount a successful infection. On the other hand, it should not be assumed that all signals to which bacteria must respond originate in the extracellular environment. For many species, even the cosseted life in a laboratory shake flask is 'stressful', in the sense that there is often a need to avoid or reverse the effects of harmful intermediates or by-products of metabolism. For example, all organisms that use dioxygen as a terminal electron acceptor have to deal with the reactive oxygen species that arise as adventitious by-products of aerobic metabolism. In bacteria, multiple protein receptors for oxygen radicals have been described, which control the expression of genes encoding enzymes that detoxify oxygen radicals or repair the damage that they cause.
Further reading: Sensory Mechanisms in Bacteria: Molecular Aspects of Signal Recognition