• FRET
• RhoGTPase
• biosensor
• fluorescent protein

• Fluorescence Resonance Energy Transfer/methods
• Biosensing Techniques/methods
• Luminescent Proteins/metabolism
• Luminescent Proteins/genetics
• Humans
• Microscopy, Fluorescence/methods
• rho GTP-Binding Proteins/metabolism

• F31 GM150215 NIGMS NIH HHS
• R35 GM136226 NIGMS NIH HHS
• R35GM136226 NIH/NIGMS
• F31GM150215 NIH/NIGMS

• Salmonella
• mutagenesis
• protein engineering
• protein secretion
• type-3 secretion

• Type III Secretion Systems/genetics
• Type III Secretion Systems/metabolism
• Bacterial Proteins/genetics
• Bacterial Proteins/metabolism
• Mutagenesis
• Salmonella enterica/genetics
• Salmonella enterica/metabolism
• Gene Library
• Salmonella typhimurium/genetics
• Salmonella typhimurium/metabolism

• CBET-2043973 National Science Foundation (NSF)
• NSF DMR-2308691 National Science Foundation (NSF)
• T32GM105538 HHS | National Institutes of Health (NIH)
• NSF | National Science Foundation Graduate Research Fellowship Program (GRFP)
• T32GM008449 HHS | National Institutes of Health (NIH)

• E. coli
• disulfide bond formation
• glutaredoxin
• oxidative stress
• thioredoxin

• R01 GM049640 NIGMS NIH HHS
• R37 GM049640 NIGMS NIH HHS

• Fluorescence intensity
• PF07598 gene family
• Soluble protein
• VM proteins
• mCherry

• 4030-20 Americas Foundation
• R01AI108276 National Institute of Allergy and Infectious Diseases

• Folding
• Misfolding
• Post-translational modifications
• Protein aggregation
• Refolding

• copper toxicity
• metal efflux pumps
• thiol oxidation

• Copper/metabolism
• Escherichia coli/metabolism
• Periplasm/metabolism
• Acetylcysteine/metabolism
• Cytoplasm/metabolism
• Bacteria/metabolism
• Oxidation-Reduction
• Transcription Factors/metabolism
• Sulfhydryl Compounds/metabolism
• Alkaline Phosphatase/genetics
• Alkaline Phosphatase/metabolism
• Disulfides/metabolism

• R01 GM049640 NIGMS NIH HHS
• R37 GM049640 NIGMS NIH HHS

• CRM197
• Escherichia coli
• SHuffle strain
• carrier protein
• protein expression
• protein solubility

• Disulfide bond formation
• Disulfide proteome
• Escherichia coli
• Kinetic modelling
• Oxidative folding
• Recombinant protein production
• Systems biology

• proteins
• isolation, separation and purification

Escherichia coli, one of the most efficient expression hosts for recombinant proteins (RPs), is widely used in chemical, medical, food and other industries. However, conventional expression strains are unable to effectively express proteins with complex structures or toxicity. The key to solving this problem is to alleviate the host burden associated with protein overproduction and to enhance the ability to accurately fold and modify RPs at high expression levels. Here, we summarize the recently developed optimization strategies for the high-level production of RPs from the two aspects of host burden and protein activity. The aim is to maximize the ability of researchers to quickly select an appropriate optimization strategy for improving the production of RPs.

• antivenom
• bioassays
• envenomation
• evolution
• genomics
• modern venomics
• spatial -omics
• toxin production
• translational research
• venom

• Animals
• Proteomics
• Research
• Snakes/genetics
• Transcriptome
• Venoms/chemistry
• Venoms/genetics

• European Cooperation in Science and Technology
• Deutsche Forschungsgemeinschaft

• Escherichia coli
• Fc engineering
• aglycosylated antibody
• bispecific antibody
• cell-free expression
• disulfide bond
• effector function
• full-length immunoglobulin
• monoclonal antibody
• semi-oxidizing cytoplasm

• oxidative stress
• peroxide
• reactive oxygen species
• redox sensors
• redox signaling
• redox systems biology
• redoxin
• synthetic biology‌

• Antioxidants
• Oxidation-Reduction
• Peroxiredoxins/metabolism
• Sulfhydryl Compounds
• Synthetic Biology

• Major histocompatibility complex
• Panning
• Phage display
• T-cell receptor
• single chain T-cell receptor

• Fipronil
• Immunoassay
• Magnetosome
• Magnetospirillum gryphiswaldense
• Nanobody

• T32 EB019941 NIBIB NIH HHS
• T32 GM133395 NIGMS NIH HHS

• ARDS
• AT2
• DPPC
• ER exit
• alveoli
• crystal structure
• lipid transfer
• lysosome-related organelle
• multilamellar
• saposin

(1) Background: S-methyl methanethiosulfonate (MMTS), a sulfur containing volatile organic compound produced by plants and bacterial species, has recently been described to be an efficient anti-oomycete agent with promising perspectives for the control of the devastating potato late blight disease caused by Phytophthora infestans. However, earlier work raised questions regarding the putative toxicity of this compound. To assess the suitability of MMTS for late blight control in the field, the present study thus aimed at evaluating the effect of MMTS on a wide range of non-target organisms in comparison to P. infestans. (2) Methods: To this end, we exposed P. infestans, as well as different pathogenic and non-pathogenic fungi, bacteria, the nematode Caenorhabditis elegans as well as the plant Arabidopsis thaliana to MMTS treatment and evaluated their response by means of in vitro assays. (3) Results: Our results showed that fungi (both mycelium and spores) tolerated MMTS better than the oomycete P. infestans, but that the compound nevertheless exhibited non-negligible toxic effects on bacteria, nematodes and plants. (4) Conclusions: We discuss the mode of action of MMTS and conclude that even though this compound might be too toxic for chemical application in the field, its strong anti-oomycete activity could still be exploited when naturally released at the site of infection by plant-associated microbes inoculated as biocontrol agents.

• Keywords: bacterial volatiles
• MMTS
• Phytophthora infestans
• biofungicide

• Cys-loop receptors
• PICK1
• cysteine mutagenesis
• intracellular domain
• nicotinic acetylcholine receptors
• α7 nAChR

• OhrR regulator
• Shewanella
• organic peroxide
• oxidative stress response
• thioredoxin

• Ester synthesis activity
• Maltose-binding protein fusion tag
• Membrane-associated lipase
• Soluble expression

• Computational Biology/methods
• Cysteine/chemistry
• Databases, Protein
• Disulfides/chemistry
• Models, Molecular
• Protein Conformation
• Proteins/chemistry
• Quantum Theory
• Thermodynamics

• 9999-NIST Intramural NIST DOC

• Crystal structure
• Oxidative stress response
• Thioredoxin
• Thioredoxin reductase

• Binding Sites
• Catalytic Domain
• Crystallography, X-Ray
• Disulfides/chemistry
• Disulfides/metabolism
• Models, Molecular
• Mutagenesis, Site-Directed
• Mutation
• NADP/metabolism
• Oxidation-Reduction
• Protein Conformation
• Staphylococcus aureus/metabolism
• Substrate Specificity
• Thioredoxin-Disulfide Reductase/chemistry
• Thioredoxin-Disulfide Reductase/genetics
• Thioredoxin-Disulfide Reductase/metabolism
• Thioredoxins/chemistry
• Thioredoxins/metabolism

• Anti-Bacterial Agents/metabolism
• Bacterial Proteins/chemistry
• Catalytic Domain
• Cell Wall/metabolism
• Glutamic Acid/metabolism
• Glutaminase/metabolism
• Glutamine/metabolism
• Lipids/chemistry
• Mycobacterium tuberculosis/metabolism
• Oxidoreductases/chemistry
• Peptidoglycan/chemistry
• Protein Domains
• Staphylococcal Infections/metabolism
• Staphylococcus aureus/metabolism
• Streptococcus pneumoniae/enzymology
• Sulfhydryl Compounds/chemistry

• Wellcome Trust
• grant 101824/Z/13/Z Wellcome Trust
• ANR-10-INSB-05-02 Agence Nationale de la Recherche (French National Research Agency)
• ANR-10-LABX-49-01 Agence Nationale de la Recherche (French National Research Agency)
• AstraZeneca IMMI 2014016

Over 50 million women are exposed to the risk of malaria during pregnancy every year. Malaria during pregnancy is a leading global cause of maternal morbidity and adverse pregnancy outcomes. Adhesion of Plasmodium falciparum-infected erythrocytes to placental chondroitin-4-sulfate (CSA) has been linked to the severe disease outcome of placental malaria. Accumulated evidence strongly supports VAR2CSA as the leading placental malaria vaccine candidate. Recombinant proteins encompassing the VAR2CSA high affinity CSA binding site have been generated, and their activity as immunogens that elicit functional (inhibitory) and cross-reactive antibodies against CSA-binding parasites assessed. The expression of His-tagged proteins was compared in four different expression systems and their capacity to bind specifically to CSA was analyzed. CHO cells and E. coli SHuffle cells were the two expression systems able to express some of the recombinant proteins in reasonable amounts. Larger analytical scale production of DBL1x-2× (3D7) and DBL3x-4ε (FCR3) best expressed in CHO and E. coli SHuffle cells were performed. Purified proteins were administered to rats either alone or adjuvanted with human approved adjuvants. Analysis of the functionality and cross-reactivity of the induced antibodies allowed us to down-select the DBL1x-2(3D7) expressed in E. coli SHuffle cells as the best antigen to be transitioned to further clinical development in order to protect future pregnant women living in malaria endemic areas against the severe clinical outcomes of placental malaria.

A mix of the right parasitic protein with the right production method has yielded a vaccine candidate for placental malaria. Primarily affecting first-time pregnant women, placental malaria is estimated to cause 200,000 infant deaths and 10,000 maternal deaths annually. In this study, led by Benoît Gamain, researchers from France’s INSERM and Germany’s European Vaccine Initiative assayed a combination of proteins designed to target and block a key pathogenic mechanism of parasite-infected red blood cells. Finding the highest performing protein, the researchers also used an Escherichia coli expression system able to replicate and fold the complex protein correctly. During tests, this protein/vector combination bested others in production qualities and immunogenicity. The team’s efforts laid the foundations for a scalable, low-cost vaccine that is currently undergoing clinical trials.

Since the introduction of recombinant protein expression in the second half of the 1970s, the growth of the biopharmaceutical field has been rapid and protein therapeutics has come to the foreground. Biophysical and structural characterisation of recombinant proteins is the essential prerequisite for their successful development and commercialisation as therapeutics. Despite the challenges, including low protein solubility and inclusion body formation, prokaryotic host systems and particularly Escherichia coli, remain the system of choice for the initial attempt of production of previously unexpressed proteins. Several different approaches have been adopted, including optimisation of growth conditions, expression in the periplasmic space of the bacterial host or co-expression of molecular chaperones, to assist correct protein folding. A very commonly employed approach is also the use of protein fusion tags that enhance protein solubility. Here, a range of experimentally tested peptide tags, which present specific advantages compared to protein fusion tags and the concluding remarks of these experiments are reviewed. Finally, a concept to design solubility-enhancing peptide tags based on a protein’s pI is suggested.

• peptide tag
• polyanionic
• polycationic
• protein fusion tag
• protein solubility
• recombinant protein expression

• Rhizomucor miehei lipase
• disulfide bond
• rational design
• thermostability

• Disulfide bond formation
• VKOR
• archaea
• membrane topology

Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, co-expression partners, purification methods, and optimization of protein solubility and stability. Hence researchers are presented with thousands of potential conditions to test. Ultimately, the subset of conditions that will be sampled depends on practical considerations and prior knowledge of the enzyme(s) being studied. We have developed a rational approach to this process. We devise a pipeline comprising in silico selection of targets and construct design, and high-throughput expression screening, target enrichment, and hit identification. We have applied this pipeline to a test set of Arabidopsis thaliana cell wall glycosyltransferases known to be challenging to obtain in soluble form, as well as to a library of cell wall glycosyltransferases from other plants including agricultural and biofuel crops. The screening results suggest that recombinant cell wall glycosyltransferases in general have a very low soluble:insoluble ratio in lysates from heterologous expression cultures, and that co-expression of chaperones as well as lysis buffer optimization can increase this ratio. We have applied the identified preferred conditions to Reversibly Glycosylated Polypeptide 1 from Arabidopsis thaliana, and processed this enzyme to near-purity in unprecedented milligram amounts. The obtained preparation of Reversibly Glycosylated Polypeptide 1 has the expected arabinopyranose mutase and autoglycosylation activities.

• Cell Wall/genetics
• Cell Wall/metabolism
• Enzyme Activation
• Gene Expression
• Glycosyltransferases/genetics
• Glycosyltransferases/isolation & purification
• Glycosyltransferases/metabolism
• High-Throughput Screening Assays
• Plant Cells/enzymology
• Recombinant Proteins/genetics
• Recombinant Proteins/isolation & purification
• Recombinant Proteins/metabolism

• U.S. Department of Energy
• Carlsbergfondet

Many animal toxins may target the same molecules that need to be controlled in certain pathologies; therefore, some toxins have led to the formulation of drugs that are presently used, and many other drugs are still under development. Nevertheless, collecting sufficient toxins from the original source might be a limiting factor in studying their biological activities. Thus, molecular biology techniques have been applied in order to obtain large amounts of recombinant toxins into Escherichia coli. However, most animal toxins are difficult to express in this system, which results in insoluble, misfolded, or unstable proteins. To solve these issues, toxins have been fused with tags that may improve protein expression, solubility, and stability. Among these tags, the SUMO (small ubiquitin-related modifier) has been shown to be very efficient and can be removed by the Ulp1 protease. However, removing SUMO is a labor- and time-consuming process. To enhance this system, here we show the construction of a bicistronic vector that allows the expression of any protein fused to both the SUMO and Ulp1 protease. In this way, after expression, Ulp1 is able to cleave SUMO and leave the protein interest-free and ready for purification. This strategy was validated through the expression of a new phospholipase D from the spider Loxosceles gaucho and a disintegrin from the Bothrops insularis snake. Both recombinant toxins showed good yield and preserved biological activities, indicating that the bicistronic vector may be a viable method to produce proteins that are difficult to express.

• Loxosceles gaucho
• SUMO tag
• construction vector
• disintegrins Ulp1 protease
• phospholipase D
• protein expression
• snake toxin
• spider toxin
• venom toxin

• C-type like domain (CTLD)
• CD93
• Disulfide bond
• DsbC
• Erv1p
• Inflammation

Escherichia coli has been widely used for the production of recombinant proteins. To improve protein production yields in E. coli, directed engineering approaches have been commonly used. However, there are only few reported examples of the isolation of E. coli protein production strains using evolutionary approaches. Here, we first give an introduction to bacterial evolution and mutagenesis to set the stage for discussing how so far selection- and screening-based approaches have been used to isolate E. coli protein production strains. Finally, we discuss how evolutionary approaches may be used in the future to isolate E. coli strains with improved protein production characteristics.

• Escherichia coli
• Evolution
• Mutagenesis
• Protein production
• Recombinant protein
• Strain isolation

• Biological Evolution
• DNA, Bacterial/metabolism
• Escherichia coli/isolation & purification
• Mutagenesis
• Mutation/genetics
• Recombinant Proteins/biosynthesis

• Stiftelsen för Strategisk Forskning (SE)
• Vetenskapsrådet
• SystemsX transition postdoc fellowship

• 2H, 13C, 15N-enriched protein production
• Native disulfide bonds
• Nuclear magnetic resonance spectroscopy
• Rhizopus chinensis lipase

• Carbon Isotopes/metabolism
• Deuterium/metabolism
• Disulfides/chemistry
• Disulfides/metabolism
• Escherichia coli/genetics
• Escherichia coli/metabolism
• Glycosylation
• Isotope Labeling
• Lipase/chemistry
• Lipase/genetics
• Lipase/metabolism
• Nitrogen Isotopes/metabolism
• Pichia/genetics
• Pichia/metabolism
• Protein Folding
• Recombinant Proteins/chemistry
• Recombinant Proteins/genetics
• Recombinant Proteins/metabolism
• Rhizopus/chemistry
• Rhizopus/enzymology

• N01AA21004 NIAAA NIH HHS
• U54 GM094597 NIGMS NIH HHS
• High-end Foreign Experts Recruitment Program
• Six Talent Peaks Project in Jiangsu Province
• 333 Project in Jiangsu Province
• NSFC
• National High Technology Research and Development Program of China (863 Program)
• 111 Project

• Computational models
• Hydrogen peroxide
• Kinetics
• Oxidative stress
• Peroxiredoxin
• Redox
• Systems biology

• Antioxidants/metabolism
• Hydrogen Peroxide/isolation & purification
• Hydrogen Peroxide/metabolism
• Kinetics
• Oxidation-Reduction
• Oxidative Stress
• Reactive Oxygen Species/chemistry
• Reactive Oxygen Species/metabolism
• Signal Transduction

Allicin (diallyl thiosulfinate) from garlic is a highly potent natural antimicrobial substance. It inhibits growth of a variety of microorganisms, among them antibiotic-resistant strains. However, the precise mode of action of allicin is unknown. Here, we show that growth inhibition of Escherichia coli during allicin exposure coincides with a depletion of the glutathione pool and S-allylmercapto modification of proteins, resulting in overall decreased total sulfhydryl levels. This is accompanied by the induction of the oxidative and heat stress response. We identified and quantified the allicin-induced modification S-allylmercaptocysteine for a set of cytoplasmic proteins by using a combination of label-free mass spectrometry and differential isotope-coded affinity tag labeling of reduced and oxidized thiol residues. Activity of isocitrate lyase AceA, an S-allylmercapto-modified candidate protein, is largely inhibited by allicin treatment in vivo. Allicin-induced protein modifications trigger protein aggregation, which largely stabilizes RpoH and thereby induces the heat stress response. At sublethal concentrations, the heat stress response is crucial to overcome allicin stress. Our results indicate that the mode of action of allicin is a combination of a decrease of glutathione levels, unfolding stress, and inactivation of crucial metabolic enzymes through S-allylmercapto modification of cysteines.

• Escherichia coli (E. coli)
• S-allylmercapto modification
• allicin
• antibiotic action
• bacteria
• disulfide
• garlic (Allium sativum)
• redox proteomics
• thiol

The human pathogen Mycobacterium tuberculosis (Mtb) likely utilizes host fatty acids as a carbon source during infection. Gluconeogenesis is essential for the conversion of fatty acids into biomass. A rate-limiting step in gluconeogenesis is the conversion of fructose 1,6-bisphosphate to fructose 6-phosphate by a fructose bisphosphatase (FBPase). The Mtb genome contains only one annotated FBPase gene, glpX. Here we show that, unexpectedly, an Mtb mutant lacking GLPX grows on gluconeogenic carbon sources and has detectable FBPase activity. We demonstrate that the Mtb genome encodes an alternative FBPase (GPM2, Rv3214) that can maintain gluconeogenesis in the absence of GLPX. Consequently, deletion of both GLPX and GPM2 is required for disruption of gluconeogenesis and attenuation of Mtb in a mouse model of infection. Our work affirms a role for gluconeogenesis in Mtb virulence and reveals previously unidentified metabolic redundancy at the FBPase-catalysed reaction step of the pathway.

Mycobacterium tuberculosis feeds on host fatty acids during infection, a process that requires a fructose bisphosphatase (FBPase) enzyme for gluconeogenesis. Here, Ganapathy et al. show that the bacterium has two different FBPases and that this enzymatic activity is required for full virulence.

• Antimicrobial properties
• Cell membrane permeability
• Royalisin
• Shortened form Royalisin

• Animals
• Anti-Bacterial Agents/chemistry
• Anti-Bacterial Agents/pharmacology
• Bees
• Fatty Acids/chemistry
• Hydrophobic and Hydrophilic Interactions
• Insect Proteins/chemistry
• Insect Proteins/pharmacology
• Intercellular Signaling Peptides and Proteins
• Microbial Sensitivity Tests
• Molecular Structure
• Permeability
• Proteins/chemistry
• Proteins/pharmacology
• Pseudomonas aeruginosa/drug effects
• Pseudomonas aeruginosa/metabolism
• Staphylococcus intermedius/drug effects
• Staphylococcus intermedius/metabolism

In vitro construction of Escherichia coli ribosomes could elucidate a deeper understanding of these complex molecular machines and make possible the production of synthetic variants with new functions. Toward this goal, we recently developed an integrated synthesis, assembly and translation (iSAT) system that allows for co-activation of ribosomal RNA (rRNA) transcription and ribosome assembly, mRNA transcription and protein translation without intact cells. Here, we discovered that macromolecular crowding and reducing agents increase overall iSAT protein synthesis; the combination of 6% w/v Ficoll 400 and 2 mM DTBA yielded approximately a five-fold increase in overall iSAT protein synthesis activity. By utilizing a fluorescent RNA aptamer, fluorescent reporter proteins and ribosome sedimentation analysis, we showed that crowding agents increase iSAT yields by enhancing translation while reducing agents increase rRNA transcription and ribosome assembly. Finally, we showed that iSAT ribosomes possess ∼70% of the protein synthesis activity of in vivo-assembled E. coli ribosomes. This work improves iSAT protein synthesis through the addition of crowding and reducing agents, provides a thorough understanding of the effect of these additives within the iSAT system and demonstrates how iSAT allows for manipulation and analysis of ribosome biogenesis in the context of an in vitro transcription-translation system.