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Journal of Chromatography. A Jun 2024Affinity tags are frequently engineered into recombinant proteins to facilitate purification. Although this technique is powerful, removal of the tag is desired because...
Affinity tags are frequently engineered into recombinant proteins to facilitate purification. Although this technique is powerful, removal of the tag is desired because the tag can interfere with biological activity and can potentially increase the immunogenicity of therapeutic proteins. Tag removal is complex, as it requires adding expensive protease enzymes. To overcome this limitation, split intein based affinity purification systems have been developed in which a C-intein tag is engineered into a protein of interest for binding to a N-intein peptide ligand fixed to a chromatographic support. Tag removal in these systems is achieved by creating an active intein-complex during protein capture, which triggers a precise self-cleavage reaction. In this work, we show applications of a new split intein system, Cytiva™ ProteinSelect™. One advantage of the new system is that the N-intein ligand can be robustly produced and conjugated to large volumes of resin for production of gram scale proteins. SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager in this work were successfully captured on the affinity resin and scaled 10-fold. Another advantage of this system is the ability to sanitize the resin with sodium hydroxide without loosing the 10-20 g/L binding capacity. Binding studies with IL-1b and IFNAR-1 ECD showed that the resin can be regenerated and sanitized for up to 50 cycles without loosing binding capacity. Additionally, after several cycles of sanitization, binding capacity was retained for the SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager. As with other split intein systems, optimization was needed to achieve ideal expression and recovery. The N-terminal amino acid sequence of the protein of interest required engineering to enable the cleavage reaction. Additionally, ensuring the stability of the C-intein tag was important to prevent premature cleavage or truncation. Controlling the hold time of the expression product and the prevention of protease activity prior to purification was needed. These results demonstrate the feasibility of the Cytiva™ ProteinSelect™ system to be used in academic and industrial research and development laboratories for the purification of novel proteins expressed in either bacterial or mammalian systems.
Topics: Inteins; Chromatography, Affinity; Humans; Recombinant Proteins; Spike Glycoprotein, Coronavirus; Recombinant Fusion Proteins; SARS-CoV-2; Interleukin-1beta
PubMed: 38669943
DOI: 10.1016/j.chroma.2024.464908 -
Drug Design, Development and Therapy 2024Pristimerin, a natural triterpenoid isolated from the plants of southern snake vine and Maidenwood in the family Weseraceae, is anti-inflammatory, insecticidal,... (Review)
Review
Pristimerin, a natural triterpenoid isolated from the plants of southern snake vine and Maidenwood in the family Weseraceae, is anti-inflammatory, insecticidal, antibacterial, and antiviral substance and has been used for its cardioprotective and antitumor effects and in osteoporosis treatment. These qualities explain Pristimerin's therapeutic effects on different types of tumors and other diseases. More and more studies have shown that pristimerin acts in a wide range of biological activities and has shown great potential in various fields of modern and Chinese medicine. While Pristimerin's wide range of pharmacological effects have been widely studied by others, our comprehensive review suggests that its mechanism of action may be through affecting fundamental cellular events, including blocking the cell cycle, inducing apoptosis and autophagy, and inhibiting cell migration and invasion, or through activating or inhibiting certain key molecules in several cell signaling pathways, including nuclear factor κB (NF-κB), phosphatidylinositol 3-kinase/protein kinase B/mammalian-targeted macromycin (PI3K/Akt/mTOR), mitogen-activated protein kinases (MAPKs), extracellular signal-regulated protein kinase 1/2 (ERK1/2), Jun amino-terminal kinase (JNK1/2/3), reactive oxygen species (ROS), wingless/integrin1 (Wnt)/β-catenin, and other signaling pathways. This paper reviews the research progress of Pristimerin's pharmacological mechanism of action in recent years to provide a theoretical basis for the molecular targeting therapy and further development and utilization of Pristimerin. It also provides insights into improved treatments and therapies for clinical patients and the need to explore pristimerin as a potential facet of treatment.
Topics: Animals; Humans; Antineoplastic Agents, Phytogenic; Apoptosis; Pentacyclic Triterpenes; Signal Transduction; Triterpenes
PubMed: 38779590
DOI: 10.2147/DDDT.S460093 -
The Journal of Biological Chemistry Sep 2023Reductive dehalogenases are corrinoid and iron-sulfur cluster-containing enzymes that catalyze the reductive removal of a halogen atom. The oxygen-sensitive and...
Reductive dehalogenases are corrinoid and iron-sulfur cluster-containing enzymes that catalyze the reductive removal of a halogen atom. The oxygen-sensitive and membrane-associated nature of the respiratory reductive dehalogenases has hindered their detailed kinetic study. In contrast, the evolutionarily related catabolic reductive dehalogenases are oxygen tolerant, with those that are naturally fused to a reductase domain with similarity to phthalate dioxygenase presenting attractive targets for further study. We present efficient heterologous expression of a self-sufficient catabolic reductive dehalogenase from Jhaorihella thermophila in Escherichia coli. Combining the use of maltose-binding protein as a solubility-enhancing tag with the btuCEDFB cobalamin uptake system affords up to 40% cobalamin occupancy and a full complement of iron-sulfur clusters. The enzyme is able to efficiently perform NADPH-dependent dehalogenation of brominated and iodinated phenolic compounds, including the flame retardant tetrabromobisphenol, under both anaerobic and aerobic conditions. NADPH consumption is tightly coupled to product formation. Surprisingly, corresponding chlorinated compounds only act as competitive inhibitors. Electron paramagnetic resonance spectroscopy reveals loss of the Co(II) signal observed in the resting state of the enzyme under steady-state conditions, suggesting accumulation of Co(I)/(III) species prior to the rate-limiting step. In vivo reductive debromination activity is readily observed, and when the enzyme is expressed in E. coli strain W, supports growth on 3-bromo-4-hydroxyphenylacetic as a sole carbon source. This demonstrates the potential for catabolic reductive dehalogenases for future application in bioremediation.
Topics: Escherichia coli; NADP; Oxygen; Vitamin B 12; Phenols; Electron Spin Resonance Spectroscopy; Hydrolases; Rhodobacteraceae; Protein Structure, Tertiary; Models, Molecular; Maltose-Binding Proteins; Recombinant Fusion Proteins; Coenzymes
PubMed: 37495113
DOI: 10.1016/j.jbc.2023.105086 -
Nature Communications May 2024The UK observed a marked increase in scarlet fever and invasive group A streptococcal infection in 2022 with severe outcomes in children and similar trends worldwide....
The UK observed a marked increase in scarlet fever and invasive group A streptococcal infection in 2022 with severe outcomes in children and similar trends worldwide. Here we report lineage M1 to be the dominant source of invasive infections in this upsurge. Compared with ancestral M1 strains, invasive M1 strains exhibit reduced genomic diversity and fewer mutations in two-component regulator genes covRS. The emergence of M1 is dated to 2008. Following a bottleneck coinciding with the COVID-19 pandemic, three emergent M1 clades underwent rapid nationwide expansion, despite lack of detection in previous years. All M1 isolates thus-far sequenced globally have a phylogenetic origin in the UK, with dispersal of the new clades in Europe. While waning immunity may promote streptococcal epidemics, the genetic features of M1 point to a fitness advantage in pathogenicity, and a striking ability to persist through population bottlenecks.
Topics: Streptococcus pyogenes; United Kingdom; Humans; Streptococcal Infections; Phylogeny; COVID-19; Pandemics; Scarlet Fever; Mutation; Repressor Proteins; SARS-CoV-2; Genome, Bacterial; Europe; Bacterial Proteins
PubMed: 38729927
DOI: 10.1038/s41467-024-47929-7 -
Marine Drugs Apr 2024Red phycoerythrin (R-PE) is a highly valuable protein found in an edible seaweed, . It is used extensively in biotechnological applications due to its strong...
Red phycoerythrin (R-PE) is a highly valuable protein found in an edible seaweed, . It is used extensively in biotechnological applications due to its strong fluorescence and stability in diverse environments. However, the current methods for extracting and purifying R-PE are costly and unsustainable. The aim of the present study was to enhance the financial viability of the process by improving the extraction and purification of R-PE from dried and to further enhance R-PE value by incorporating it into a tandem dye for molecular biology applications. A combination of ultrafiltration, ion exchange chromatography, and gel filtration yielded concentrated (1 mg·mL) R-PE at 99% purity. Using purified PE and Cyanine5 (Cy5), an organic tandem dye, phycoerythrin-Cy5 (PE-Cy5), was subsequently established. In comparison to a commercially available tandem dye, PE-Cy5 exhibited 202.3% stronger fluorescence, rendering it suitable for imaging and analyzes that require high sensitivity, enhanced signal-to-noise ratio, broad dynamic range, or shorter exposure times to minimize potential damage to samples. The techno-economic analysis confirmed the financial feasibility of the innovative technique for the extraction and purification of R-PE and PE-Cy5 production.
Topics: Phycoerythrin; Carbocyanines; Seaweed; Fluorescent Dyes; Chromatography, Ion Exchange; Chromatography, Gel; Ultrafiltration; Rhodophyta; Pigments, Biological; Edible Seaweeds; Porphyra
PubMed: 38786588
DOI: 10.3390/md22050197 -
Emerging Microbes & Infections Dec 2023Dermatophytic pseudomycetoma is a rare invasive infection, involving both immunocompetent and immunocompromised individuals. Since the discovery of inherited immune...
Dermatophytic pseudomycetoma is a rare invasive infection, involving both immunocompetent and immunocompromised individuals. Since the discovery of inherited immune disorders such as the impairment of gene, extended dermatophyte infections are mostly ascribed to any of these host factors. This study is to present and explore the potential causes in a fatal dermatophytic pseudomycetoma patient. We present a chronic and deep pseudomycetoma caused by the common dermatophyte which ultimately led to the death of the patient. Mycological examination, genetic studies and host immune responses against fungi were performed to explore the potential factors. The patient had decreased lymphocyte counts with significantly reduced CD4 T cells, although all currently known genetic parameters proved to be normal. Through functional studies, we demonstrated that peripheral blood mononuclear cells from the patient showed severe impairment of adaptive cytokine production upon fungus-specific stimulation, whereas innate immune responses were partially defective. This is, to our knowledge, the first report of fatal dermatophytic pseudomycetoma in a patient with non-HIV CD4 lymphocytopenia, which highlights the importance of screening for immune deficiencies in patients with deep dermatophytosis.
Topics: Humans; Dermatomycoses; Mycetoma; Female; Middle Aged; Microsporum; Fatal Outcome; Caspase 9; Receptors, Interleukin-7; Mutation; Rare Diseases; CD4 Lymphocyte Count; Immunity, Innate
PubMed: 37128909
DOI: 10.1080/22221751.2023.2208685 -
Protein Expression and Purification Jul 2023The ever-increasing speed of biotherapeutic drug discovery has driven the development of automated and high throughput purification capabilities. Typically, purification...
The ever-increasing speed of biotherapeutic drug discovery has driven the development of automated and high throughput purification capabilities. Typically, purification systems require complex flow paths or third-party components that are not found on a standard fast protein liquid chromatography instrument (FPLC) (e.g., Cytiva's ÄKTA) to enable higher throughput. In early mAb discovery there is often a trade-off between throughput and scale where a high-throughput process requires miniaturized workflows necessitating a sacrifice in the amount of material generated. At the interface of discovery and development, flexible automated systems are required that can perform purifications in a high-throughput manner, while also generating sufficient quantities of preclinical material for biophysical, developability, and preclinical animal studies. In this study we highlight the engineering efforts to generate a highly versatile purification system capable of balancing the purification requirements between throughput, chromatographic versatility, and overall product yields. We incorporated a 150 mL Superloop into an ÄKTA FPLC system to expand our existing purification capabilities. This allowed us to perform a range of automated two-step tandem purifications including primary affinity captures (protein A (ProA)/immobilized metal affinity chromatography (IMAC)/antibody fragment (Fab)) followed by secondary polishing with either size exclusion (SEC) or cation exchange (CEX) chromatography. We also integrated a 96 deep-well plate fraction collector into the ÄKTA FPLC system with purified protein fractions being analyzed by a plate based high performance liquid chromatography instrument (HPLC). This streamlined automated purification workflow allowed us to process up to 14 samples within 24 h, enabling purification of ∼1100 proteins, monoclonal antibodies (mAbs), and mAb related protein scaffolds during a 12-month period. We purified a broad range of cell culture supernatant volumes, between 0.1 and 2 L, with final purification yields up to 2 g. The implementation of this new automated, streamlined protein purification process greatly expanded our sample throughput and purification versatility while also enabling the accelerated production of greater quantities of biotherapeutic candidates for preclinical in vivo animal studies and developability assessment.
Topics: Animals; Antibodies, Monoclonal; Chromatography, Affinity; Chromatography, High Pressure Liquid; Staphylococcal Protein A; Drug Discovery
PubMed: 37023994
DOI: 10.1016/j.pep.2023.106269 -
Infectious Diseases Now Jun 2024Athletes are vulnerable to Staphylococcus aureus infections due to skin-to-skin contact and skin abrasions during training and competitions involving sharied sport... (Review)
Review
Athletes are vulnerable to Staphylococcus aureus infections due to skin-to-skin contact and skin abrasions during training and competitions involving sharied sport equipment or toiletries, which promote the spread of the bacteria between athletes and within sport teams. This results not only in higher prevalence of S.aureus carriage among athletes compared to the general population, but also in outbreaks of infections, particularly skin infections, within sports teams. To limit the spread of S. aureus among athletes, a decolonization protocol can be applied when clustered cases of S. aureus infections occur, especially if Panton-Valentine leukocidin-producing strains are implicated. Finally, to avoid exposing athletes to S.aureus transmission/colonization, it is recommended to establish strict and clearly formulated individual and collective hygiene rules and to regularly disinfect shared sports equipment.
Topics: Humans; Staphylococcus aureus; Staphylococcal Infections; Athletes; Sports; Carrier State; Paris; Bacterial Toxins; Leukocidins; Exotoxins; Prevalence; Hygiene; Sports Equipment; Anniversaries and Special Events; Disease Outbreaks
PubMed: 38849255
DOI: 10.1016/j.idnow.2024.104882 -
Journal of Agricultural and Food... Jun 2024This review aims to provide an updated overview of the effects of protein extraction/recovery on antinutritional factors (ANFs) in plant protein ingredients, such as... (Review)
Review
This review aims to provide an updated overview of the effects of protein extraction/recovery on antinutritional factors (ANFs) in plant protein ingredients, such as protein-rich fractions, protein concentrates, and isolates. ANFs mainly include lectins, trypsin inhibitors, phytic acid, phenolic compounds, oxalates, saponins, tannins, and cyanogenic glycosides. The current technologies used to recover proteins (e.g., wet extraction, dry fractionation) and novel technologies (e.g., membrane processing) are included in this review. The mechanisms involved during protein extraction/recovery that may enhance or decrease the ANF content in plant protein ingredients are discussed. However, studies on the effects of protein extraction/recovery on specific ANFs are still scarce, especially for novel technologies such as ultrasound- and microwave-assisted extraction and membrane processing. Although the negative effects of ANFs on protein digestibility and the overall absorption of plant proteins and other nutrients are a health concern, it is also important to highlight the potential positive effects of ANFs. This is particularly relevant given the rise of novel protein ingredients in the market and the potential presence or absence of these factors and their effects on consumers' health.
Topics: Animals; Chemical Fractionation; Nutritive Value; Plant Proteins; Trypsin Inhibitors; Humans
PubMed: 38780067
DOI: 10.1021/acs.jafc.4c00380 -
Scientific Reports Apr 2024Green, photosynthesizing plants can be proficiently used as cost-effective, single-use, fully biodegradable bioreactors for environmentally-friendly production of a...
Green, photosynthesizing plants can be proficiently used as cost-effective, single-use, fully biodegradable bioreactors for environmentally-friendly production of a variety of valuable recombinant proteins. Being near-infinitely scalable and most energy-efficient in generating biomass, plants represent profoundly valid alternatives to conventionally used stationary fermenters. To validate this, we produced a plastome-engineered tobacco bioreactor line expressing a recombinant variant of the protein A from Staphylococcus aureus, an affinity ligand widely useful in antibody purification processes, reaching accumulation levels up to ~ 250 mg per 1 kg of fresh leaf biomass. Chromatography resin manufactured from photosynthetically-sourced recombinant protein A ligand conjugated to agarose beads demonstrated the innate pH-driven ability to bind and elute IgG-type antibodies and allowed one-step efficient purification of functional monoclonal antibodies from the supernatants of the producing hybridomas. The results of this study emphasize the versatility of plant-based recombinant protein production and illustrate its vast potential in reducing the cost of diverse biotechnological applications, particularly the downstream processing and purification of monoclonal antibodies.
Topics: Staphylococcal Protein A; Ligands; Plants, Genetically Modified; Chromatography; Recombinant Proteins; Antibodies, Monoclonal; Immunoglobulin G; Plant Proteins; Chromatography, Affinity
PubMed: 38622266
DOI: 10.1038/s41598-024-59266-2