Simple Purification Research Articles

Intein Based Fusion Proteins: Great Tags for the Soluble Production and Convenient Purification of Recombinant Proteins.

The main problem in the recombinant protein expression in E. coli strains, especially for high-yield production, is the accumulation in un-folded and inactive inclusion bodies. A suitable solution is the direction into the soluble cytoplasmic products by solubilizing tags. The use of inteins with self-cleaving ability, in addition to increase the chance of soluble protein expression, facilitates their purification process. In this review article, papers related to the use of intein tags for soluble expression or protein purification were collected regardless the time limit. Available databases including Pubmed, google scholar, ScienceDirect, Web of Science, Scopus, and Embase was searched. The best condition for soluble expression or purification was focused in all articles. There are various intein tags commercially available in expression vectors that results in gaining our goal in facilitating the recombinant protein solubilization as well as its simple purification. It is enough to induce the self-cleavage property of the intein, which varies according to the type of intein used. In this way, the target protein is easily separated from the purification tag without the need to add protease enzymes such as enterokinase or treatment with various chemicals. The most common affinity tag in intein-based systems is Chitin Binding Domain attached to the chitin resin. In this review article, we introduced proteins or peptides which produced in fusion to intein tags and discussed about their expression condition and purification process in order to enhance the chance of soluble expression and intein cleavage in a single stage, respectively.

Isolation of Cardanol Fractions from Cashew Nutshell Liquid (CNSL): A Sustainable Approach

Exploring sustainable approaches to replace petroleum-based chemicals is an ongoing challenge in reducing the carbon footprint. Due to the complexity and percentage variation in nature-generated molecules, which further varies based on geographical origin and the purification protocol adopted, a better isolation strategy for individual components is required. Agrowaste from the cashew industry generates phenolic lipid (cardanol)-rich cashew nutshell liquid (CNSL) and has recently shown extensive commercial utility. Cardanol naturally exists as a mixture of three structurally different components with C15-alkylene chains: monoene, diene, and triene. The separation of these three fractions has been a bottleneck and is crucial for certain structural designs and reproducibility. Herein, we describe the gram-scale purification of cardanol into each component using flash column chromatography within the sustainability framework. The solvent used for elution is recovered and reused after each stage (up to 82%), making it a cost-effective and sustainable purification strategy. This simple purification technique replaces the alternative high-temperature vacuum distillation, which requires substantial energy consumption and poses vacuum fluctuation and maintenance challenges. Three components (monoene 42%, diene 22%, and triene 36%) were isolated with good purity and were fully characterized by 1H and 13C NMR, GC-MS, HPLC, and FTIR spectroscopy. The present work demonstrates that greener and simpler strategies pave the way for the isolation of constituents from nature-sourced biochemicals and unleash the potential of CNSL-derived fractions for high-end applications.

Construction of highly hydrophilic PAN-co-IA/CNTs@TA composite membrane with large flux and high retention for purification of dye wastewater

The rising pollution caused by dye industrial wastewater has led to irreversible destruction of the environment and human health. Hence, there is an urgent need to construct hydrophilic separation membranes possessing high flux and retention to be used in water treatment. To solve this problem, polyacrylonitrile-co-itaconic acid (PAN-co-IA) copolymer was prepared as matrix, tannic acid (TA) encapsulated carbon nanotubes (CNTs) named as CNTs@TA was used as nano-modifier to design the PAN-co-IA/CNTs@TA composite flat membrane by the non-solvent-induced phase separation technique. The copolymerization of IA improved the hydrophilicity of PAN, in addition, the hydrogen bonding formation between IA and TA on the surface of CNTs ensured the stable existence of CNTs@TA particles in the composite membrane. At the same time, the π-π bonds between TA and CNTs avoided the loss of TA during the wastewater separation process. The introduction of IA, TA, and CNTs endowed the PAN-co-IA/CNTs@TA composite membrane with excellent hydrophilicity and double-layer finger pore structure, which achieved its high pure water flux of 3102.63 Lm-2h-1. What's more, the PAN-co-IA/CNTs@TA composite membrane behaved large flux (>1600 Lm-2h-1) and high retention (>97 %) for various cationic dye wastewater based on the electrostatic adsorption mechanism. In addition, the high hydrophilicity and the reinforcing effect of CNTs endowed PAN-co-IA/CNTs@TA membrane with excellent cycling stability. Therefore, this research provides a simple and convenient dye wastewater purification strategy with promising application prospects.

Synthesis of 3‐Arylazoindoles through Iron(III)‐Catalyzed C−H Azolation of Indoles and Arylhydrazines

AbstractA variety of functionalized 3‐arylazoindoles were prepared in good to excellent yields through an iron(III)‐catalyzed C−H azolation of 2‐substituted indoles with arylhydrazines under mild reaction conditions. The reaction underwent an initial TBN‐mediated nitrosation, 1,5‐hydrogen migration, intramolecular condensation, and 1,5‐hydrogen migration over four steps in one pot. A paracyclophane‐derived 3‐phenylazoindole could be prepared in 25% yield over three steps and some prepared 3‐arylazoindoles showed a different fluorescence emission wavelength and existed obvious pH conditions response. The present method highlights broad substrate scope of easily available starting materials, wide range of functional group compatibility, gram scalable preparations, high atom economy, avoidance of unstable and explosive azolation reagents, and simple purification operations by extraction, washing, and recrystallization without flash column chromatography.

A simple graphene oxide-based DNA purification strategy for plant pathogen detection.

The on-site molecular detection of plant pathogens is particularly important for the development of sustainable agriculture. Extracting DNA from plant tissues, microbes or coexisting environments is complex, labor-intensive and time-consuming. To facilitate this process, we propose a DNA purification strategy based on graphene oxide (GO). The excellent adsorption ability of GO was verified by visualizing changes in its microscopic surface and macroscopic mixture. To further optimize the DNA purification, we determined the optimal GO concentration and treatment time at 95 °C (2 mg mL-1 and 2 min, respectively). We confirmed that our strategy is effective on plant tissues and various microorganisms, and that the obtained DNA can be directly used for polymerase chain reaction amplification. Combining the proposed GO-based DNA purification method with the loop-mediated isothermal amplification method is superior, in terms of the required steps, time, cost and detection effect, to the cetyltrimethylammonium bromide method and a commercial kit for detecting plant pathogens. We present a feasible, rapid, simple and low-cost DNA purification method with high practical value for scientific applications in plant pathogen detection. This strategy can also provide important technical support for future research on plant-microbial microenvironments. © 2024 Society of Chemical Industry.

Multicomponent Synthesis of α-Branched Amines via a Zinc-Mediated Carbonyl Alkylative Amination Reaction.

Methods for the synthesis of α-branched alkylamines are important due to their ubiquity in biologically active molecules. Despite the development of many methods for amine preparation, C(sp3)-rich nitrogen-containing compounds continue to pose challenges for synthesis. While carbonyl reductive amination (CRA) between ketones and alkylamines is the cornerstone method for α-branched alkylamine synthesis, it is sometimes limited by the sterically demanding condensation step between dialkyl ketones and amines and the more restricted availability of ketones compared to aldehydes. We recently reported a "higher-order" variant of this transformation, carbonyl alkylative amination (CAA), which utilized a halogen atom transfer (XAT)-mediated radical mechanism, enabling the streamlined synthesis of complex α-branched alkylamines. Despite the efficacy of this visible-light-driven approach, it displayed scalability issues, and competitive reductive amination was a problem for certain substrate classes, limiting applicability. Here, we report a change in the reaction regime that expands the CAA platform through the realization of an extremely broad zinc-mediated CAA reaction. This new strategy enabled elimination of competitive CRA, simplified purification, and improved reaction scope. Furthermore, this new reaction harnessed carboxylic acid derivatives as alkyl donors and facilitated the synthesis of α-trialkyl tertiary amines, which cannot be accessed via CRA. This Zn-mediated CAA reaction can be carried out at a variety of scales, from a 10 μmol setup in microtiter plates enabling high-throughput experimentation, to the gram-scale synthesis of medicinally-relevant compounds. We believe that this transformation enables robust, efficient, and economical access to α-branched alkylamines and provides a viable alternative to the current benchmark methods.

Improvement in Dibenzofuran-Based Hole Transport Materials for Flexible Perovskite Solar Cells.

The π-conjugated system and the steric configuration of hole transport materials (HTMs) could greatly affect their various properties and the corresponding perovskite solar cells' efficiencies. Here, a molecular engineering strategy of incorporating different amounts of p-methoxyaniline-substituted dibenzofurans as π bridge into HTMs was proposed to develop oligomer HTMs, named mDBF, bDBF, and tDBF. Upon extending the π-conjugation of HTMs, their HOMO energy levels were slightly deepened, significantly increasing the thermal stability and hole mobility. The incorporation of p-methoxyaniline bridges built one or two additional triphenylamine propeller structures, resulting in a denser film. Here, the tDBF-based n-i-p flexible perovskite solar cells createdchampion efficiency, giving a power conversion efficiency of 19.46%. And the simple synthesis and purification process of tDBF contributed to its low manufacturing cost in the laboratory. This work provided a reference for the development of low-cost and efficient HTMs.

Bacterial cellulose as an ideal potential treatment for burn wounds: A comprehensive review.

Burn wound regeneration is a complex process, which has many serious challenges such as slow wound healing, secondary infection, and inflammation. Therefore, it is essential to utilise appropriate biomaterials to accelerate and guide the wound healing process. Bacterial cellulose (BC), a natural polymer synthesised by some bacteria, has attracted much attention for wound healing applications due to its unique properties including excellent physicochemical and mechanical properties, simple purification process, three-dimensional (3D) network structure similar to extracellular matrix, high purity, high water holding capacity and significant permeability to gas and liquid. BC's lack of antibacterial activity significantly limits its biomedical and tissue engineering application, but adding antimicrobial agents to it remarkably improves its performance in tissue regeneration applications. Burn wound healing is a complex long-lasting process. Using biomaterials in wound treatment has shown that they can satisfactorily accelerate wound healing. The purpose of this review is to elaborate on the importance of BC-based structures as one of the most widely used modern wound dressings in the treatment of burn wounds. In addition, the combination of various drugs, agents, cells and biomolecules with BC to expand its application in burn injury regeneration is discussed. Finally, the main challenges and future development direction of BC-based structures for burn wound repair are considered. The four most popular search engines PubMed/MEDLINE, Science Direct, Scopus and Google Scholar were used to help us find relevant papers. The most frequently used keywords were bacterial cellulose, BC-based biocomposite, wound healing, burn wound and vascular graft.

Evidence for long-term efficacy of a membrane filtration device in rural villages in Ghana

Drinking water contaminated by pathogenic micro-organisms increases the risk of infectious gastrointestinal disease which could potentially lead to acute kidney injury and even death, particularly amongst the young and the elderly. Earlier studies have shown a substantial reduction in the incidence of diarrheal disease over a period of one year using a polysulfone membrane water gravity-powered water filtration device. The current report is a continuation of these studies to assess the long-term effects of the innovative method on diarrheal incidence rates over a 4-year follow-up period. This follow-up study monitored the trend of self-reported diarrheal events in all households in the previously studied villages for 5 months, in the last half of each study year, using the same questionnaire utilized in the earlier study. Three villages that had no device yet installed served as controls. We computed monthly diarrheal incidence rates for all study years (standardized to per 100 person-months) and compared these to the pre-device incidence rate in 2018 and in the control group, using the Wilcoxon rank sum exact test. The average diarrheal incidence rates of 1.5 p100pm in 2019, 2.19 p100pm in 2021, and 0.54p100pm in 2022 were significantly different from an earlier study that reported 17.8 p100pm rates before the devices were installed in 2018, (all p-values < 0.05). Concomitantly, self-reported diarrheal infections were substantially higher in the “control villages” not yet having the filtration device installed (80.9, 77.6, and 21.5 per 100 pm). The consistent and large reduction in diarrhea incidence documents the long-term efficacy of the use of the membrane filtration device. This simple water purification method using gravity flow improves public health in remote regions with limited resources.

A simple water purification tool as an effort to meet clean water needs

Water is necessary for all types of life on Earth. Water is also essential for daily activities, such as cooking, washing dishes and dirty clothes, and cleansing oneself. Water is scientifically constituted of numerous components despite its appearance of being clear and odorless. Among these numerous applications, bathing, washing clothing, and defecating account for almost 85% of total water usage. According to Water Aid 2016, more than 40% of the population in 16 nations lacked access to water facilities and wells. These impoverished groups must gather water from ponds and rivers and spend a significant portion of their daily income on safe drinking water. As a result, this study was carried out to perform simple filtering of clothing convection factory waste utilizing a simple water filtration method. This study used experimental research methods. The pretest-posttest design (pretest-posttest design) was employed in the study. The convection manufacturing wastewater was sampled in Semanggi Village, Pasar Kliwon, Surakarta City. The investigation findings revealed that the BOD and COD indicators remained below the quality criteria. After processing, the quality of liquid waste has improved and satisfied quality criteria. TSS (37.5), pH (1.08), BOD (5.4), and phosphate (9.2) efficiency all decreased. Meanwhile, the COD parameter climbed in value but remained inside the quality standard’s lower limit; therefore, it had no significance for efficacy.

Identification, characterization and application of M16AT, a new organic solvent-tolerant (R)-enantio-selective type IV amine transaminase from Mycobacterium sp. ACS1612.

Biocatalysis has emerged as a powerful alternative to traditional chemical methods, especially for asymmetric synthesis. As biocatalysts usually exhibit excellent chemical, regio- and enantioselectivity, they facilitate and simplify many chemical processes for the production of a broad range of products. Here, a new biocatalyst called, R-selective amine transaminases (R-ATAs), was obtained from Mycobacterium sp. ACS1612 (M16AT) using in-silico prediction combined with a genome and protein database. A two-step simple purification process could yield a high concentration of pure enzyme, suggesting that industrial application would be inexpensive. Additionally, the newly identified and characterized R-ATAs displayed a broad substrate spectrum and strong tolerance to organic solvents. Moreover, the synthetic applicability of M16AT has been demonstrated by the asymmetric synthesis of (R)-fendiline from of (R)-1-phenylethan-1-amine.

Threading the needle: Achieving simplicity and performance in cellulose alkanoate ω-carboxyalkanoates for amorphous solid dispersion

Most active pharmaceutical ingredients (APIs) suffer from poor water solubility, often keeping them from reaching patients. To overcome the issues of poor drug solubility and subsequent low bioavailability, amorphous solid dispersions (ASDs) have garnered much attention. Cellulose ester derivatives are of interest for ASD applications as they are benign, sustainable-based, and successful in commercial drug delivery systems, e.g. in osmotic pump systems and as commercial ASD polymers. Synthesis of carboxy-pendant cellulose esters is a challenge, due in part to competing reactions between carboxyls and hydroxyls, forming ester crosslinks. Herein we demonstrate proof-of-concept for a scalable synthetic route to simple, yet highly promising ASD polymers by esterifying cellulose polymers through ring-opening of cyclic succinic or glutaric anhydride. We describe the complexity of such ring-opening reactions, not previously well-described, and report ways to avoid gelation. We report synthesis, characterization, and preliminary in vitro ASD evaluations of fifteen such derivatives. Synthetic routes were designed to accommodate these criteria: no protecting groups, no metal catalysts, mild conditions with standard reagents, simple purification, and one-pot synthesis. Finally, these designed ASD polymers included members that maintained fast-crystallizing felodipine in solution and release it from an ASD at rather high 20 % drug loading (DL).

Expression and purification of truncated recombinant human angiotensin converting enzyme 2 (trhACE2) capable of binding spike protein of SARS-CoV-2

SARS-CoV-2 infects the host through the interaction be-tween spike protein and the ACE2 (Angiotensin convert-ing enzyme 2) receptor on the host cell surface. There-fore, ACE2 is considered as a key target in drug devel-opment against COVID-19. In this study, we generated the truncated recombinant human ACE2 (trhACE2) expressed on Escherichia coli and evaluated its binding activity to spike protein of SARS-CoV-2. The sequence of ACE2 (18-119 aa) was cloned into pET28a(+) plasmid and transformed into E. coli DH5α strain and finally was transferred to E. coli BL21 (DE3) cells for protein expression. Protein trhACE2 was purified by affinity chromatography using a HisTrap HP column. Protein purity was above 95% and production yield was 75 mg/l. The purified protein was refolded by dialysis method. trhACE2 was evaluated for its ability to bind the spike protein by sandwich ELISA assay and surface plasmon resonance (SPR). The results showed that trhACE2 has the ability to bind spike protein of SARS-CoV-2 with the equilibrium dissociation constant Kd=15.7 nM. With a relatively simple and inexpensive expression and purifi-cation process on E. coli system, there is the potential to develop trhACE2 for preventive and supportive thera-pies against COVID-19.

Multiplexable and Scalable Aqueous Synthesis Platform for Oleate-Based, Bilayer-Coated Gold Nanoparticles.

Despite gold-based nanomaterials having a unique role in nanomedicine, among other fields, synthesis limitations relating to reaction scale-up and control result in prohibitively high gold nanoparticle costs. In this work, a new preparation procedure for lipid bilayer-coated gold nanoparticles in water is presented, using sodium oleate as reductant and capping agent. The seed-free synthesis not only allows for size precision (8-30nm) but also remarkable particle concentration (10mm Au). These reaction efficiencies allow for multiplexing and reaction standardization in 96-well plates using conventional thermocyclers, in addition to simple particle purification via microcentrifugation. Such a multiplexing approach also enables detailed spectroscopic investigation of the nonlinear growth process and dynamic sodium oleate/oleic acid self-assembly. In addition to scalability (at gram-level), resulting gold nanoparticles are stable at physiological pH, in common cell culture media, and are autoclavable. To demonstrate the versatility and applicability of the reported method, a robust ligand exchange with thiolated polyethylene glycol analogues is also presented.

Exploiting Orthogonal C-C Cross-Coupling Reactions for Chemistry-on-the-Complex: Modular Assembly of 2,6-Di(quinolin-8-yl)pyridine Ruthenium(II) Photosensitizer Triads.

In this work, we present a concise modular assembly strategy using one universal heteroleptic 2,6-di(quinolin-8-yl)pyridine-based ruthenium(II) complex as a starting building block. Extending the concept from established ligand modifications and subsequent complexation (classical route), the later appearing chemistry-on-the-complex methodology was used for late-stage syntheses, i.e., assembling discrete building blocks to molecular architectures (here: dyad and triads). We focused on Suzuki-Miyaura and Sonogashira cross-couplings as two of the best-known C-C bond forming reactions. Both were performed on one building block complex bearing a bromine and TIPS-protected alkyne for functional group interconversion (bromine to TMS-protected alkyne, a benzyl azide, or a boronic acid pinacol ester moiety with ≥95% isolated yield and simple purification) as well as building block assemblies using both a triarylamine-based donor and a naphthalene diimide-based acceptor in up to 86% isolated yield. Additionally, the developed purification via automated flash chromatography is simple compared to tedious manual chromatography for ruthenium(II)-based substrates in the classical route. Based on the preliminary characterization by steady-state spectroscopy, the observed emission quenching in the triad (55%) serves as an entry to rationally optimize the modular units via chemistry-on-the-complex to elucidate energy and electron transfer.

Coprecipitation as a One-Step Se Separation for Determination of Isotope Ratios Completed with Revised Uncertainty Evaluation.

This study introduces a simplified purification method for analyzing 82Se/78Se isotope ratios in diverse natural samples using hydride generation MC-ICP-MS. Unlike the thiol resin method, which is time-consuming and sensitive to the concentrations of reagents used at individual stages, our proposed alternative is quicker, simpler, and robust. The procedure involves coprecipitation of selenium with iron hydroxide and dissolution in hydrochloric acid. Combining hydride generation and a second cleanup stage achieves sufficient purification for Se isotope ratio measurements. The method is efficient, taking 3-4 h after sample decomposition, utilizing common reagents [HCl, Fe(NO3)3, NH4Cl] without evaporation or clean lab conditions. Results on 82Se/78Se isotope ratios in various matrices are presented, comparing them with literature data. All isotopic results have been subjected to a newly proposed state-of-the-art approach to uncertainty estimation dedicated to isotope ratio measurements. The approach is based on applying Monte Carlo simulations with consideration of different samples' results normalized by the expected value. By doing that, we obtained estimated uncertainty for any Se sample with the influence of particular measurements on the final estimation included. We employ a Monte Carlo simulation-based uncertainty estimation approach for isotope ratio measurements, providing estimated uncertainty for each selenium sample.

Wheat flour-derived amyloid fibrils for efficient removal of organic dyes from contaminated water

Amyloid fibrils derived from different proteins have been proved as a promising material for adsorption of various pollutants from wastewater, which showed advantages of low cost and eco-friendliness. However, most of the amyloid fibrils derived from animal-based proteins with high environmental footprint, while more sustainable amyloid fibrils derived from plant materials are desirable. In this study, a plant-derived amyloid fibril was extracted from the commonly used wheat flour with a simple and scalable protein purification and fibrillization process. Interestingly, the amyloid fibrils showed good adsorption capacity towards typical organic dyes (Eosin Y (EY) and Congo red (CR)) from contaminated water. Adsorption kinetic analysis indicated the adsorption process to EY or CR by wheat flour amyloid well fitted with a pseudo-second-order model. The adsorption also followed a Langmuir isothermal model with adsorption capacities of 333 mg/g and 138 mg/g towards CR and EY, respectively. This work demonstrated the feasibility to utilize the plant-based amyloid fibril for organic dyes removal from contaminated water, which provided an affordable, sustainable and scalable tool for organic dyes removal from wastewater.Graphical

DIPEA‐Assisted Efficient One‐Pot Three‐Component Synthesis of Novel β‐Carboline Tethered Pyran Derivatives

AbstractA potential DIPEA‐assisted approach has been unfolded for the synthesis of novel β‐carboline tethered‐4H‐pyran derivatives via one‐pot condensation of 1‐formyl‐9H‐β‐carbolines, β‐keto esters and malononitrile in ethanol at ambient temperature. A library of 28 new β‐carboline tethered 4H‐pyran derivatives has been developed. The significant features of the present strategy include high efficiency, excellent yield, inexpensive catalyst, mild reaction conditions, multicomponent character of reaction and simple purification procedure.

Determination of Eugenol Residues in Fish Tissue, Transport, and Temporary Water of Aquatic Product by Gas Chromatography-Tandem Mass Spectrometry with Application of the Electrospun Nanofibrous Membrane.

Using gas chromatography-tandem mass spectrometry and electrospun nanofibrous membrane, we developed and validated a simple, rapid, and sensitive methodology for quantifying eugenol residues in fish tissue and water samples. Fish tissue extract and water samples (315 samples) collected from three southeastern China provinces (Shanghai, Zhejiang, and Fujian), originating from eight provinces of Zhejiang, Jiangsu, Shandong, Guangdong, Fujian, Anhui, Shanghai, and Jiangxi, from April 2021 to April 2023 were filtered with an electrospun nanofiber membrane, extracted with trichloromethane/n-hexane, and directly concentrated to dry after simple purification. An internal standard of p-terphenyl in n-hexane and 5-µL injection volumes of the solutions was used to analyze eugenol via internal calibration with a minimum concentration of 0.5 µg/L in water samples and 0.1 µg/kg in aquatic product samples. The highest amount of eugenol was detected in Fujian province, possibly due to the higher temperature during transportation, while the lowest amount was found in Shanghai, which mainly uses temporary fish-culture devices. This is a fast, inexpensive, and effective method for testing large quantities of fish water and meat samples.

Sustainable Synthesis of Indole-Substituted Densely Functionalized Pyrrole.

A novel chromatography- and catalyst-free methodology has been developed for the synthesis of poly substituted pyrrole in good yields via a multicomponent reaction of arylglyoxal, 1,3-dicarbonyl, indole, and aromatic amine. This strategy provides various advantages such as simple experimental and workup procedures, mild reaction conditions, no added catalyst, use of green solvent, and simple purification procedure of pure product without using column chromatography. This green method offers a simple and highly effective strategy to synthesize a wide range of indole-pyrrole conjugates in a one-pot operation.