Anion-exchange Chromatography Research Articles

Fructan Catabolism by Rumen Microbiota of Cattle and Sheep

Fructans serve as the primary form of storage carbohydrate in cool-season grasses, but little is known about potential differences in ruminal fermentation of fructans between cattle and sheep. An ex vivo study was conducted to evaluate species differences in fructan catabolism. Buffered media containing ground orchardgrass (Dactylis glomerata L.) substrate was inoculated with uncultivated rumen microbiota obtained from cattle and sheep (n = 4 species−1). Fructan profiles were monitored over the incubation period (8 h; 39 °C) using high-performance anion-exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD). In both species, disappearance of long-chain fructans (degree of polymerization [DP] > 8) was evident by 2 h of incubation (p < 0.01), whereas short-chain fructans (DP 4–8) increased from 0 to 2 h prior to subsequent degradation (p < 0.01). However, the overall rate of long-chain fructan catabolism was greater in bovine versus ovine fermentations, particularly between 2 and 4 h (p < 0.01). Additionally, rapid utilization of short-chain fructans occurred from 2 to 4 h in bovine fermentations, but was delayed in ovine fermentations, with substantial degradation occurring only after 4 h of incubation (p < 0.01). These results indicate that rumen microbiota of cattle may have a greater capacity for fructan degradation.

Acid hydrolysis conditions for quantification of meningococcal X polysaccharide in a pentavalent vaccine using HPAEC-PAD/ESI-MS

A selective and sensitive method was evaluated for quantitation of meningococcal X (Men X) polysaccharide in pentavalent meningococcal A, C, W, Y and X conjugate vaccine using different acid hydrolysis conditions like HCl, TFA, HF, HF-TFA, and HF–HCl. High-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using CarboPac PA10 column was used to identify the hydrolyzed products based on retention time and its comparison with monosaccharide standards. Complete release of glucosamine (GlcN) from Men X in monovalent bulk and pentavalent vaccine samples was achieved using HF hydrolysis at 80 °C for 2 h. The Men X HF-hydrolyzed polysaccharide to glucosamine along with the reference standard was identified using collision-induced dissociation (CID) electrospray mass spectroscopy and the MS/MS fragments of m/z 162, m/z 144 and m/z 84. Meningococcal polysaccharide concentration was determined with a correlation coefficient r2 >0.99 using polysaccharide reference standard. The serogroups A, W, and Y were converted to their monosaccharides units and quantified using this method however, milder acid hydrolysis 0.1 M HCl 80 °C 2 h for release of sialic acid for Men C polysaccharide was found to be more suitable. These methods will provide necessary tools and prove to be beneficial to laboratories developing new saccharide-based vaccine combinations.

Immunomodulatory activity and protective effect of a capsular polysaccharide in Caenorhabditis elegans, isolated from Lactobacillus fermentum GBJ

A capsular polysaccharide, namely CPS-2, was isolated from Lactobacillus fermentum GBJ, purified using DEAE-52 anion exchange chromatography, and structurally characterized. We found that CPS-2 is homogenous, has an average molecular weight of 377 KDa, and is mainly composed of galactose and glucose at a molar ratio of 1.54:1.00. Its backbone comprises α-D-Galp-(1 → 3), α-D-Galp-(1 → 3,6), β-D-Glcp-(1 → 2), β-D-Galp-(1 → 6), and α-D-Galp-(1 → 4) residues with a side chain of β-D-Glcp-(1→). CPS-2 exerts an immunomodulatory effect by improving the proliferation and phagocytosis of macrophage RAW264.7 and promoting the secretion of NO and cytokines. The maximum secretion levels of IL-1β, IL-6, IL-10, and TNF-α were 1.96-, 0.11-, 0.22-, and 0.46-fold higher than those of the control, respectively. Furthermore, CPS-2 could significantly enhance the antioxidant system, extend lifespan, and improve stress tolerance of Caenorhabditis elegans at both exposure doses of 31.25 and 62.5 μg/mL. The average lifespan of nematodes reached a maximum in the 62.5 μg/mL-treated group after 10.39 days, 6.56 h, and 23.56 h in normal, oxidative stress, and heat shock environment, with extension percentages of 16.61 %, 43.23 %, and 15.77 %, respectively; therefore, CPS-2 displays an anti-aging effect. The significant bioactivity of CPS-2 promotes its application as a promising immunomodulatory and anti-aging ingredient in the food or pharmaceutical field.

Gastric carboxylesterases of the edible crab Cancer pagurus (Crustacea, Decapoda) can hydrolyze biodegradable plastics

A promising strategy to counteract the progressing plastic pollution of the environment can involve the replacement of persistent plastics with biodegradable materials. Biodegradable polymers are enzymatically degradable by various hydrolytic enzymes. However, these materials can reach the environment in the same way as conventional plastics. Therefore, they are accessible to terrestrial, freshwater, and marine biota. Once ingested by marine organisms, highly active enzymes in their digestive tracts may break down biodegradable compounds. We incubated microparticles of five different biodegradable plastics, based on polylactictic acid (PLA), polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT) and polyhydroxybutyrate-co-valerate (PHBV), in-vitro with the gastric fluid of the edible crab Cancer pagurus and evaluated the hydrolysis rates by pH Stat titration. A plastic blend of PLA with PBAT showed the highest hydrolysis rate. The enzymes in the gastric fluid of crabs were separated by anion exchange chromatography. Fractions with carboxylesterase activity were identified using fluorescent methylumbelliferyl (MUF)-derivatives. Pooled fractions with high carboxylesterase activity also hydrolyzed a PLA/PBAT plastic blend. Carboxylesterases showed molecular masses of 40–45 kDa as determined by native gel electrophoresis (SDS-PAGE). Our study demonstrated that digestive carboxylesterases in the gastric fluid of C. pagurus exhibit a high potential for hydrolyzing certain biodegradable plastics. Since esterases are common in the digestive tract of organisms, it seems likely that other invertebrates possess the ability to hydrolyze biodegradable plastics.

ISOLATION AND STUDY OF POLYSACCHARIDES OF MARGILAN RADISH RAPHANUS SATIVUS

The purpose of this research is to isolate and study the physicochemical characteristics of polysaccharides in the seeds of the Margilan radish of the Brassicaceae family cultivated in the Republic of Uzbekistan.
 For the first time, a water-soluble fraction of polysaccharides was isolated from radish seeds by sowing Margilan successive extraction after the extraction of proteins and polyphenols. Anion exchange chromatography and gel filtration were used to obtain homogeneous polysaccharides. The polysaccharide fraction was separated by ion-exchange chromatography, purified on a Sephadex G-100 column. As a result, two polysaccharides RSP1 and RSP2 were obtained. The monosaccharide composition of the isolated polysaccharides was established. Analysis of monosaccharide residues showed that the composition of the RSP1 polysaccharide is represented by monosaccharides in the following composition: rhamnose - 0.7%, ribose - 4.7%, arabinose - 55.6%, xylose - 1.5%, mannose - 3.7%, glucose - 5.1% and galactose - 28.7%. The composition of the RSP2 polysaccharide is represented by monosaccharides: ribose - 2.8%, arabinose - 9.4%, mannose - 0.9%, glucose - 64.9% and galactose - 18.4%. From the obtained results, it can be seen that the RSP1 polysaccharide consists mainly of arabinose (55.6%) and galactose (28.7%) residues and belongs to the type of arabinogalactans, and the RSP2 polysaccharide mainly consists of glucose residues (64.9%) and galactose (18.4%).

In vitro α-Amylase and α-Glucosidase Inhibitory Activity of Dioscorin from Dioscorea alata

Dioscorea alata is a species of yam found in many countries in Southeast Asia. It contains many bioactive compounds consisting of steroidal saponins, phenolic compounds flavonoids, polysaccharides, and proteins. Eighty to eighty-five of the soluble proteins in the tuber of D. alata are dioscorin. Dioscorin (31 kDa) is the major storage protein of yam tuber, exhibiting biochemical and immunomodulatory properties, such as antioxidant activity, reducing blood pressure and angiotensin I converting enzyme (ACE) inhibitors activity. The aims of this research focused on extraction of the total soluble protein from D. alata tuber, purification of dioscorin, and study of in vitro inhibitory activity of α-amylase and α-glucosidase of dioscorin extracts. The results showed that a maximum yield of total soluble protein was obtained from the buffers extraction using Tris-HCl buffer at pH 9.0 resulting in the protein concentration of 15.10 mg/g with a protein extraction yield of 31.56 ± 2.36%. The crude protein was further recovered using ammonium sulfate precipitation and dialysis. SDS-PAGE analysis showed that dioscorin from crude extracts was precipitated at the concentration of ammonium sulfate of 60% saturation. The purified dioscorin was obtained from anion exchange chromatography (DEAE-Sephacel) and its molecular weight of 31 kDa was estimated on SDS-PAGE. Furthermore, the α-amylase inhibitory activities of crude extract (1 mg/mL), purified dioscorin obtain from gastric digestion and purified dioscorin obtained from intestinal digestion were at 14.44% 2.21% and 78.13%, respectively. Moreover, the results showed that the α-glucosidase inhibitory activity of the crude extract, and purified dioscorin obtained from gastric digestion were at 10.56% and 2.61%, respectively.

Purification of linearized template plasmid DNA decreases double-stranded RNA formation during IVT reaction.

After the COVID-19 pandemic, messenger RNA (mRNA) has revolutionized traditional vaccine manufacturing. With the increasing number of RNA-based therapeutics, valuable new scientific insights into these molecules have emerged. One fascinating area of study is the formation of double-stranded RNA (dsRNA) during in vitro transcription (IVT) which is considered a significant impurity, as it has been identified as a major trigger in the cellular immune response pathway. Therefore, there is a growing importance placed to develop and optimize purification processes for the removal of this by-product. Traditionally, efforts have primarily focused on mRNA purification after IVT through chromatographic separations, with anion exchange and reverse phase chromatography emerging as effective tools for this purpose. However, to the best of our knowledge, the influence and significance of the quality of the linearized plasmid have not been thoroughly investigated. Plasmids production involves the growth of bacterial cultures, bacterial harvesting and lysis, and multiple filtration steps for plasmid DNA purification. The inherent complexity of these molecules, along with the multitude of purification steps involved in their processing, including the subsequent linearization and the less-developed purification techniques for linearized plasmids, often result in inconsistent batches with limited control over by-products such as dsRNA. This study aims to demonstrate how the purification process employed for linearized plasmids can impact the formation of dsRNA. Several techniques for the purification of linearized plasmids based on both, resin filtration and chromatographic separations, have been studied. As a result of that, we have optimized a chromatographic method for purifying linearized plasmids using monolithic columns with C4 chemistry (butyl chains located in the surface of the particles), which has proven successful for mRNAs of various sizes. This chromatographic separation facilitates the generation of homogeneous linearized plasmids, leading to mRNA batches with lower levels of dsRNA during subsequent IVT processes. This finding reveals that dsRNA formation is influenced not only by RNA polymerase and IVT conditions but also by the quality of the linearized template. The results suggest that plasmid impurities may contribute to the production of dsRNA by providing additional templates that can be transcribed into sequences that anneal with the mRNA molecules. This highlights the importance of considering the quality of plasmid purification in relation to dsRNA generation during transcription. Further investigation is needed to fully understand the mechanisms and implications of plasmid-derived dsRNA. This discovery could shift the focus in mRNA vaccine production, placing more emphasis on the purification of linearized plasmids and potentially saving, in some instances, a purification step for mRNA following IVT.

Separation of Plasmid DNA Topological Forms, Messenger RNA, and Lipid Nanoparticle Aggregates Using an Ultrawide Pore Size Exclusion Chromatography Column.

Health authorities have highlighted the need to determine oligonucleotide aggregates. However, existing technologies have limitations that have prevented the reliable analysis of size variants for large nucleic acids and lipid nanoparticles (LNPs). In this work, nucleic acid and LNP aggregation was examined using prototype, low adsorption ultrawide pore size exclusion chromatography (SEC) columns. A preliminary study was conducted to determine the column's physicochemical properties. A large difference in aggregate content (17.8 vs 59.7 %) was found for a model messenger RNA (mRNA) produced by different manufacturers. We further investigated the nature of the aggregates via a heat treatment. Interestingly, thermal stress irreversibly decreased the amount of aggregates from 59.7 to 4.1% and increased the main peak area 3.3-fold. To the best of our knowledge, for the first time, plasmid DNA topological forms and multimers were separated by analytical SEC. The degradation trends were compared to the data obtained with an anion exchange chromatography method. Finally, unconjugated and fragment antigen-binding (Fab)-guided LNPs were analyzed and their elution times were plotted against their sizes as measured by DLS. Multi-angle light scattering (MALS) was coupled to SEC in order to gain further insights on large species eluting before the LNPs, which were later identified as self-associating LNPs. This study demonstrated the utility of ultrawide pore SEC columns in characterizing the size variants of large nucleic acid therapeutics and LNPs.

Adeno-associated viral capsid stability on anion exchange chromatography column and its impact on empty and full capsid separation

Recombinant adeno-associated viral vector (rAAV) mediated gene therapy is gaining traction in treating genetic disorders. Current rAAV production systems yield a mixture of capsids largely devoid of the transgene (empty capsid) compared with the desired therapeutic product (full capsid). Anion exchange chromatography (AEX) is an attractive method for separating empty and full AAV capsids because of its scalability. Resin types and buffer composition are key considerations for AEX and must support capsid stability to be suitable for downstream processing. We examined the impact of binding durations (0-8 h) using various binding ionic strengths (15-75mM), pH (7.5-9.0), resin chemistry (POROS XQ, POROS HQ, POROS I, and BIA QA monolith), and proprietary Q resins with different ligand densities for effects on capsid stability. Empty capsids were altered upon extended binding, leading to retention time shifts and loss of resolution between empty and full capsids. Viral capsid protein analysis reveals that full capsids have more viral capsid protein 3 (VP3) proteins than empty capsids. Analytical hydrophilic liquid chromatography showed that empty capsid retention time shift is accompanied by changes to the empty capsid's native VP3 protein. Among the potential stabilizing additives considered, magnesium chloride was the most effective at reducing negative impacts caused by extended binding.

Structural characterization and adjuvant action of Paulownia tomentosa flower polysaccharide on the immune responses to classical swine fever vaccine in mice.

Paulownia tomentosa flower polysaccharide (PTFP) from dried cultured P. tomentosa flowers, is widely known for its immunomodulatory activities. Here, PTFP was extracted from Paulownia tomentosa flower using hot water extraction, followed by ethanol precipitation methods. Structural characterization of PTFP was revealed by scanning electron microscope, high-performance anion-exchange chromatography, gel chromatography, ultraviolet and infrared spectral. Meanwhile, adjuvant action of PTFT on the immune responses to classical swine fever vaccine in mice was evaluated to further proclaim the immune regulatory effect of PTFP. The results showed that PTFP was a type of heteropolysaccharide with a dense, rough surface and high molecular weight (667.02 kDa), mainly composed of glucose (30.93%), rhamnose (29.99%), galactose (15.66%), arabinose (6.95%), mannose (5.52%), and xylose (4.80%). The results of gel chromatography suggested that the molecular configuration of PTFP may be a spherical structure. The infrared spectrum results confirmed that the functional groups and chemical bond of PTFP contained -OH, O-H, C-H, C=O, C-O, etc. Moreover, PTFP exhibited obvious immune enhancement effect by improving concanavalin A (ConA), lipopolysaccharide (LPS), and CSFV E2-stimulated splenocyte growth and natural killer cell activity in CSFV-immunized mice. Similarly, the titers of CSFV E2-specific IgG, IgG1, IgG2a, and IgG2b antibodies and IFN-γ and IL-10 levels in CSFV-immunized mice were distinctly increased by PTFP treatment. Overall, PTFP was a macromolecular heteropolysaccharide primarily containing glucose and rhamnose, and possessed the auxiliary effect of immune enhancement on the immune responses to classical swine fever vaccine.

Self-assembling Gn head ferritin nanoparticle vaccine provides full protection from lethal challenge of Dabie bandavirus in aged ferrets.

Dabie bandavirus (DBV) is an emerging tick-borne virus that causes severe fever with thrombocytopenia syndrome (SFTS) in infected patients. Human SFTS symptoms progress from fever, fatigue, and muscle pain to the depletion of white blood cells and platelets with fatality rates up to 30%. The recent spread of its vector tick to over 20 states in the United States increases the potential for outbreaks of the SFTS beyond the East Asia. Thus, the development of vaccine to control this rapidly emerging virus is a high priority. In this study, we applied self-assembling ferritin (FT) nanoparticle to enhance the immunogenicity of DBV Gn head domain (GnH) as a vaccine target. Mice immunized with the GnH-FT nanoparticle vaccine induced potent antibody responses and cellular immunity. Immunized aged ferrets were fully protected from the lethal challenge of DBV. Our study describes the GnH-FT nanoparticle vaccine candidate that provides protective immunity against the emerging DBV infection.

A cell-free multi-enzyme cascade for the synthesis of CDP-glycerol.

CDP-glycerol is a nucleotide-diphosphate-activated version of glycerol. In nature, it is required for the biosynthesis of teichoic acid in Gram-positive bacteria, which is an appealing target epitope for the development of new vaccines. Here, a cell-free multi-enzyme cascade was developed to synthetize nucleotide-activated glycerol from the inexpensive and readily available substrates cytidine and glycerol. The cascade comprises five recombinant enzymes expressed in E. coli that were purified by immobilized metal affinity chromatography. As part of the cascade, ATP is in-situ regenerated from polyphosphate to reduce synthesis costs. The enzymatic cascade was characterized at laboratory scale and the products analyzed by high performance anion exchange chromatography (HPAEC)-UV and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). After the successful synthesis was confirmed, a design of experiments approach was used to screen for optimal operation conditions (temperature, pH value and MgCl2 concentration). Overall, a substrate conversion of 89% was achieved with respect to the substrate cytidine.

Bacterial cellulose-based submicrobundle framework-induced hyperporous cellulose microspheres functionalized by ionic liquids for anion exchange chromatography

Ovalbumin (OVA) is widely used in food and pharmaceutical industries and can be separated using ion exchange chromatography. However, both rapid adsorption kinetics and great adsorption capacity are impeded by the nanoscale porous structures of chromatography media. Here, hyperporous cellulose microspheres (BCMs) with well-controllable micron-sized macropores are fabricated by a facile one-step pre-crosslinking strategy. Bacterial cellulose nanofibers and dissolved cellulose chains are pre-crosslinked by epichlorohydrin to form submicrobundles and generate an interconnected gel network. The preliminary gelation blocks induce a hyperporous framework during the recrystallization and assembly of regenerated cellulose. BCMs have demonstrated decreased mass transfer resistance and increased accessible active sites due to the micron-sized convective macropores (2.04 μm) and high specific surface area (194.25 m2 g−1). Cationic imidazolium ILs (AMIBr) functionalized BCMs (IL-BCMs) are prepared as anion exchangers. IL-BCMs exhibit rapid adsorption kinetics for OVA, reaching 85% of the equilibrium adsorption amount within 30 min. Additionally, IL-BCMs demonstrate endothermic chemosorption, high adsorption capacity (150.30 mg g−1, 25 °C), reusability (>85% removal efficiency after seven cycles), and selectivity towards OVA at pH 6.0. Continuous fixed-bed adsorption further suggests the practicality of IL-BCMs in ion exchange chromatography, indicating that IL-BCMs are appealing media for protein separation and purification.

Plasma measurements of the dual sugar test reveal carbohydrate immediately alleviates intestinal permeability caused by exertional heat stress.

The aim of this set of randomised cross-over studies was to determine the impact of progressive heat exposure and carbohydrate or protein feeding during exertional stress on small intestine permeability using a dual sugar test. In our previous work, and typically in the field, recovery of lactulose and l-rhamnose is measured cumulatively in urine. This follow-up study exploits our novel high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) protocol to accurately quantify the sugars in plasma. Endurance-trained participants completed experimental trial A (ET-A; n=8), consisting of 2h running at 60% in temperate, warm and hot ambient conditions, and/or experimental trial B (ET-B; n=9), consisting of 2h running at 60% in the heat while consuming water, carbohydrate or protein. Blood samples were collected and plasma lactulose (L) and l-rhamnose (R) appearance, after dual sugar solution ingestion at 90min of exercise, was quantified by HPAEC-PAD to measure plasma L/R and reveal new information about intestinal permeability immediately post-exercise and during recovery. In ET-A, plasma L/R increased immediately post-exercise in hot compared with temperate and warm conditions, while, in ET-B, carbohydrate alleviated this, and this information was otherwise missed when measuring urine L/R. Consuming carbohydrate or protein before and during exercise attenuated small intestine permeability throughout recovery from exertional heat stress. We recommend using the dual sugar test with quantification of plasma sugars by HPAEC-PAD at intervals to maximise intestinal permeability data collection in exercise gastroenterology research, as this gives additional information compared to urinary measurements. KEY POINTS: Intestinal permeability is typically assessed using a dual sugar test, by administering a drink containing non-metabolisable sugars (e.g. lactulose (L) and l-rhamnose (R)) that can enter the circulation by paracellular translocation when the epithelium is compromised, and are subsequently measured in urine. We demonstrate that our recently developed ion chromatography protocol can be used to accurately quantify the L/R ratio in plasma, and that measuring L/R in plasma collected at intervals during the post-exercise recovery period reveals novel acute response information compared to measuring 5-h cumulative urine L/R. We confirm that exercising in hot ambient conditions increases intestinal epithelial permeability immediately after exercise, while consuming carbohydrate or protein immediately before and during exercise attenuates this. We recommend using our dual sugar absorption test protocol to maximise intestinal epithelial permeability data collection in exercise gastroenterology research and beyond.

Ultrasound-assisted extraction and characterization of Penthorum chinense polysaccharide with anti-inflammatory effects

Penthorum chinense has been used in both food and medication for many years, and polysaccharide of which was considered as one of the bioactive compounds. However, the extraction process of polysaccharide from P. chinense (PCP) was not well optimized. Ultrasound-assisted extractionhas been widely employed in the extraction of natural products for its compliance with the concept of green and economic chemistry. To better investigate the structure and biology activity of PCP, response surface methodology was employed to optimize the ultrasound-assisted extraction conditions of PCP. The optimum extraction for the ultrasound-assisted extraction of PCP were obtained as ratio of solvent to material 40 mL/g, ultrasonic power 380 W, and extraction time of 50 min. The yield of PCP reached 8.71% under these optimized conditions. PCP was further purified by using anion exchange chromatography and gel filtration, an acidic fraction PCP-AP-1 was hereby obtained. The results of structural elucidation indicated that PCP-AP-1 was a typical pectic polysaccharide with a molecular weight of 66360 Da, mainly composed of galacturonic acid (68.5 mol%), followed by arabinose (9.8 mol%), rhamnose (9.4 mol%), glucose (7.7 mol%), with homogalacturonan region and rhamnogalacturonan I regions. In vitro study showed that PCP-AP-1 could improve the inflammation induced by lipopolysaccharide in intestinal epithelial cells, which was probably performed through the inhibition of multiple signaling pathways including the inhibition of TLR4, NOD1/2 and NF-κB pathway, as well as the reduction of NLRP3 inflammasome. This study defined the type of polysaccharide present in P. chinense and revealed a potential of application this plant in the prevention of intestinal inflammatory diseases.

Analytical Ultracentrifugation-Calibrated Anion-Exchange Chromatography for Sensitive and Intact Determination of Osteopontin in Infant Formula and Dairy Products.

Osteopontin is a crucial protein ingredient that has been applied in fortified dairy products and infant formula. It has great significance to infant gut health and brain development. However, current techniques including enzyme-linked immunosorbent assay and liquid chromatography coupled with mass spectrometry are still facing the bottleneck of low sensitivity and indirect quantification. Moreover, the unavailable certified commercial OPN standard hinders its accurate quantification. Herein, a novel method of anion-exchange chromatography was established to determine OPN concentration in several dairy matrices. The polarity-reversed capillary isoelectric focusing was utilized to measure the exact isoelectric point (pI) to support method development for OPN separation. Analytical ultracentrifugation was used to calibrate the purity of intact OPN to develop an in-house reference standard. The method showed the merits of limits of detection to 0.04 mg/100 g, relative standard deviation of reproducibility <5% for 13 out of 14 tested matrices, and an average recovery rate of 101.3%. This method has shown the potential to be adopted as an international standard method for the quantification of intact OPN in infant formula and dairy products.

A Versatile and Efficient Method to Isolate DNA-Polymer Conjugates.

We present a facile and adaptable method to purify and isolate DNA-polymer conjugates from different uncharged homo, random, or block copolymer families. Anion exchange chromatography is used to separate the reaction solution and retrieve the excess unreacted polymer and oligonucleotide. The stationary phase has a high efficiency (25 nmol of DNA per run), facilitating the purification of large batches without compromising the peak shape and resolution. To demonstrate the versatility of this method, different types of polymers, including acrylates, methacrylates, and acrylamides containing hydrophilic and hydrophobic blocks, were purified with high yields. Additionally, DNA-polymer conjugates with various DNA block lengths were also successfully purified, further highlighting the broad applicability of this method.

A fast protein liquid chromatography method for purification of myoglobin from different species

The aim of this study is to describe a fast method for the purification of high-purity myoglobin for Mass Spectroscopy analyses and to use it as standard-grade material. A three-step Fast Protein Liquid Chromatography (FPLC) method was used to produce high-purity myoglobin. SEC 650 gel filtration followed by an Enrich Q anion exchange chromatography was used to produce myoglobin in acceptable purity for most research methods. A second filtration step was carried out by narrow field SEC 70 gel to prepare high-purity myoglobin at standard grade purity and capable of Mass Spectroscopy analyses. At least 90% pure myoglobin was obtained by applying two chromatography steps in the samples of three species, and over 99% pure myoglobin was obtained in standard material quality and suitable for mass spectroscopy when the additional narrow field SEC 70 chromatography step was applied. The proposed method provides higher purity compared to other methods and can be applied in a shorter time. FPLC columns significantly reduce the duration of the chromatography steps. At the same time, the use of solid extraction columns instead of dialysis reduced the long overnight dialysis process to a few minutes.

Purification and fractionation of phycobiliproteins from Arthrospira platensis and Corallina officinalis with evaluating their biological activities

Phycobiliproteins (PBPs) are a class of water-soluble pigments with a variety of biological functions that are present in red macroalgae and cyanobacterial species. The crude forms of phycocyanin (C-PC) from the blue green alga Arthrospira platensis and allophycocyanin (APC) from the red macroalga Corallina officinalis were extracted and purified by ammonium sulphate precipitation, anion exchange chromatography, and size exclusion chromatography methods, respectively. The obtained C-PC and APC from A. platensis and C. officinalis were 0.31 mg/mL and 0.08 mg/mL, respectively, with molecular masses of “17.0 KDa and 19.0 KDa” and “15.0 KDa and 17.0 KDa” corresponding to α and β subunits, respectively. FT-IR was used to characterize the purified APC and C-PC in order to look into their structures. Highly purified extracts (A620/A280 > 4.0) were obtained from subtractions’ PC3 and PC4 that were tested for their biological activities. APC and C-PC crude extracts plus their fractions exhibited potent anti-oxidant in different ratios by using three techniques. PC1 showed high anti-inflammatory (75.99 and 74.55%) and anti-arthritic (78.89 and 76.92%) activities for C. officinalis and A. platensis, respectively compared with standard drugs (72.02 and 71.5%). The methanolic and water extracts of both species showed greater antibacterial efficacy against Gram +ve than Gram −ve marine bacteria. Our study shed light on the potential medical uses of C-PC and APC extracted from the tested species as natural substances in a variety of foods and drugs. Further investigations are required to explore the diverse chemical natures of distinct PBPs from different cyanobacteria and red algae because their amino acid sequences vary among different algal species.

Murine model identifies tropomyosin as IgE cross-reactive protein between house dust mite and coho salmon that possibly contributes to the development of salmon allergy.

Recently, we have developed a method to identify IgE cross-reactive allergens. However, the mechanism by which IgE cross-reactive allergens cause food allergy is not yet fully understood how. In this study, we aimed to understand the underlying pathogenesis by identifying food allergens that cross-react with house dust mite allergens in a murine model. Allergenic protein microarray analysis was conducted using serum from mice intraperitoneally injected with Dermatophagoides pteronyssinus (Der p) extract plus alum or alum alone as controls. Der p, Dermatophagoides farinae (Der f), coho salmon extract-sensitized and control mice were analyzed. Serum levels of IgE against Der p, Der f, coho salmon extract, protein fractions of coho salmon extract separated by ammonium sulfate precipitation and anion exchange chromatography, and recombinant coho salmon tropomyosin or actin were measured by an enzyme-linked immunosorbent assay. A murine model of cutaneous anaphylaxis or oral allergy syndrome (OAS) was established in Der p extract-sensitized mice stimulated with coho salmon extract, tropomyosin, or actin. Protein microarray analysis showed that coho salmon-derived proteins were highly bound to serum IgE in Der p extract-sensitized mice. Serum IgE from Der p or Der f extract-sensitized mice was bound to coho salmon extract, whereas serum IgE from coho salmon extract-sensitized mice was bound to Der p or Der f extract. Analysis of the murine model showed that cutaneous anaphylaxis and oral allergic reaction were evident in Der p extract-sensitized mice stimulated by coho salmon extract. Serum IgE from Der p or Der f extract-sensitized mice was bound strongly to protein fractions separated by anion exchange chromatography of coho salmon proteins precipitated with 50% ammonium sulfate, which massively contained the approximately 38 kDa protein. We found that serum IgE from Der p extract-sensitized mice was bound to recombinant coho salmon tropomyosin. Der p extract-sensitized mice exhibited cutaneous anaphylaxis in response to coho salmon tropomyosin. Our results showed IgE cross-reactivity of tropomyosin between Dermatophagoides and coho salmon which illustrates salmon allergy following sensitization with the house dust mite Dermatophagoides. Our method for identifying IgE cross-reactive allergens will help understand the underlying mechanisms of food allergies.