Anion-exchange Chromatography Research Articles

Maximal performance of intact N-glycopeptide enrichment using sequential HILIC and MAX columns.

Low abundance and heterogeneity of N-glycosylation at the peptide level poses a great challenge to the structural and functional analysis of glycosylation in the field of glycobiology. Solving this conundrum requires a sufficient and specific method for intact N-glycopeptide enrichment. Using the C18 or HLB desalting column followed by the mixed-mode strong anion exchange (MAX) or hydrophilic interaction chromatography (HILIC) glycopeptide enrichment column are commonly applied approaches for sample preparation of intact N-glycopeptides from complex samples. Herein, we compared the effects of different combinations of two desalting columns and two enrichment columns using equal amounts of mouse brain tissues from the same source. The results revealed the C18 column was a bit superior to the HLB column, and the MAX and HILIC columns were complementary on intact N-glycopeptides enrichment. Additionally, the results also demonstrated that enriching glycopeptides using a HILIC column followed by a MAX column from the flow-through solution got a better enrichment performance than the reversed order. Based on these results, the sequential enrichment of glycopeptides using HILIC and then MAX columns could maximize the enrichment performance of intact N-glycopeptides, and therefore is an option for in-depth analysis of site-specific glycoproteome.

Antioxidant, Collagenase Inhibitory, and Antibacterial Effects of Bioactive Peptides Derived from Enzymatic Hydrolysate of Ulva australis.

The protein extract of Ulva australis hydrolyzed with Alcalase and Flavourzyme was found to have multi-functional properties, including total antioxidant capacity (TAC), collagenase inhibitory, and antibacterial activities. The #5 fraction (SP5) and #7 fraction (SP7) of U. australis hydrolysate from cation-exchange chromatography displayed significantly high TAC, collagenase inhibitory, and antibacterial effects against Propionibacterium acnes, and only the Q3 fraction from anion-exchange chromatography displayed high multi-functional activities. Eight of 42 peptides identified by MALDI-TOF/MS and Q-TOF/MS/MS were selected from the results for screening with molecular docking on target proteins and were then synthesized. Thr-Gly-Thr-Trp (TGTW) displayed ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] radical scavenging activity. The effect of TAC as Trolox equivalence was dependent on the concentration of TGTW. Asn-Arg-Asp-Tyr (NRDY) and Arg-Asp-Arg-Phe (RDRF) exhibited collagenase inhibitory activity, which increased according to the increase in concentration, and their IC50 values were 0.95 mM and 0.84 mM, respectively. Peptides RDRF and His-Ala-Val-Tyr (HAVY) displayed anti-P. Acnes effects, with IC50 values of 8.57 mM and 13.23 mM, respectively. These results suggest that the U. australis hydrolysate could be a resource for the application of effective nutraceuticals and cosmetics.

High serum mannose in colorectal cancer: a novel biomarker of lymph node metastasis and poor prognosis.

Lymph node status is an important prognostic indicator and it significantly influences treatment decisions for colorectal cancer (CRC). The objective of this study was to evaluate the ability of serum monosaccharides in predicting lymph node metastasis (LNM) and prognosis. High performance anion exchange chromatography coupled with pulsed amperometric detector (HPAEC-PAD) was used to quantify serum monosaccharides from 252 CRC patients. Receiver operating characteristic (ROC) curves were used to evaluate predictive performance of parameters. Predictors of LNM were evaluated by univariate and multivariate analyses. The prognostic role of the factors was evaluated by survival analysis. The levels of serum mannose (Man) and galactose (Gal) were significantly increased in patients with LNM (p <0.0001, p =0.0017, respectively). The area under the curves (AUCs) of Man was 0.8140, which was higher than carcinoembryonic antigen (CEA) (AUC =0.6523). Univariate and multivariate analyses demonstrated histologic grade (G3) (odds ratio [OR] =2.60, p =0.043), histologic grade (mucin-producing subtype) (odds ratio [OR] =3.38, p =0.032), lymphovascular invasion (LVI) (OR =2.42, p <0.01), CEA (>5ng/ml) (OR =1.85, p =0.042) and high Man (OR =2.65, p =0.006) to be independent risk factors of LNM. The survival analysis showed that the high serum Man was independent risk factor for poor prognosis in CRC patients (HR=1.75, p =0.004). The Man is superior to CEA in prediction of LNM for CRC patients. Man is expected to be a predictor for LNM in CRC. High serum Man is associated with poor prognosis of CRC patients.

Potential Application of Yeast Cell Wall Biopolymers as Probiotic Encapsulants.

Biopolymers of yeast cell walls, such as β-glucan, mannoprotein, and chitin, may serve as viable encapsulants for probiotics. Due to its thermal stability, β-glucan is a suitable cryoprotectant for probiotic microorganisms during freeze-drying. Mannoprotein has been shown to increase the adhesion of probiotic microorganisms to intestinal epithelial cells. Typically, chitin is utilized in the form of its derivatives, particularly chitosan, which is derived via deacetylation. Brewery waste has shown potential as a source of β-glucan that can be optimally extracted through thermolysis and sonication to yield up to 14% β-glucan, which can then be processed with protease and spray drying to achieve utmost purity. While laminarinase and sodium deodecyle sulfate were used to isolate and extract mannoproteins and glucanase was used to purify them, hexadecyltrimethylammonium bromide precipitation was used to improve the amount of purified mannoproteins to 7.25 percent. The maximum chitin yield of 2.4% was attained by continuing the acid-alkali reaction procedure, which was then followed by dialysis and lyophilization. Separation and purification of yeast cell wall biopolymers via diethylaminoethyl (DEAE) anion exchange chromatography can be used to increase the purity of β-glucan, whose purity in turn can also be increased using concanavalin-A chromatography based on the glucan/mannan ratio. In the meantime, mannoproteins can be purified via affinity chromatography that can be combined with zymolase treatment. Then, dialysis can be continued to obtain chitin with high purity. β-glucans, mannoproteins, and chitosan-derived yeast cell walls have been shown to promote the survival of probiotic microorganisms in the digestive tract. In addition, the prebiotic activity of β-glucans and mannoproteins can combine with microorganisms to form synbiotics.

Preferential exclusion chromatography as a capture step for extracellular AAV harvest from adherent and suspension productions.

Preferential exclusion chromatography (PXC) sometimes described as hydrophobic interaction chromatography is a well-known, but not widely used technique for purification of Adeno-associated viruses. It employs high molarity of preferentially excluded cosolvent (salt in our case). The downside of this method is that high molarity of salt can lead to aggregation and precipitation of different compounds from the sample. In the case of viruses that are excreted to medium, the concentration of impurities is much lower compared to cell lysates, and PXC can be used as a first chromatographic, serotype independent step to concentrate and purify adeno-associated virus (AAV). Here, we explored PXC for adherent and suspension harvests using monolithic chromatographic columns (CIMmultus). Suspension extracellular adeno-associated virus, serotype 9 (AAV9) harvest had more impurities compared to adherent harvest, therefore it required higher input regarding method development. Final conditions for suspension harvest included higher molarity of binding salt and using more open channel format of chromatographic column (6µm channel size). Vector genome analysis with droplet digital polymerase chain reaction (ddPCR) revealed 84% and 97% recovery for suspension and adherent AAV9 harvest, respectively. After PXC capture step, adherent AAV9 was purified by already described ion exchange techniques. Overall process vector genome recovery, from clarified harvest to anion exchange elution fraction, was 54% measured by ddPCR. Residual host cell DNA was measured at 40ng per 1E13 vector genome, and empty AAV was below 5% in final anion exchange chromatography fraction.

Estimation of alginate purity and M/G ratio by methanolysis coupled with anion exchange chromatography

Alginates are industrially relevant polysaccharides widely used in the food and biomedical industries for their excellent gelling properties. The growing emphasis on the valorization of marine resources has evidenced the need for alternative methods for the determination of both alginate content and the M/G ratio. This study describes the application of acid methanolysis and separation by anion exchange chromatography. Five samples, including alginates extracted from Saccharina latissima, Ascophyllum nodosum, a certified standard, and two poly-uronates (Poly-M and Poly-G), were analysed for their M/G ratio and alginate content at different treatment conditions, and compared with other conventionally used or reference methods (NMR, FTIR, and colorimetric methods). Quantitative estimation of alginate was relatively accurate at optimum conditions (4 h at 100 °C), as compared with the certified standard or with other colorimetric methods. M/G ratios were not significantly different from those determined after the reference method (1H NMR) or compared to FTIR protocols. The results evidence that methanolysis may be applied to simultaneously estimate the purity and M/G ratio of alginate-rich samples in a single analysis.

Heterologous expression and characterization of a MoAA16 polysaccharide monooxygenase from the rice blast fungus Magnaporthe oryzae

BackgroundCellulose is an organic carbon source that can be used as a sustainable alternative for energy, materials, and chemicals. However, the substantial challenge of converting it into soluble sugars remains a major obstacle in its use as a biofuel and chemical feedstock. A new class of enzymes knowns as copper-dependent polysaccharide monooxygenases (PMOs) or lytic polysaccharide monooxygenases (LPMOs) can break down polysaccharides such as cellulose, chitin, and starch through oxidation. This process enhances the efficiency of cellulose degradation by cellulase. ResultsThe genome of the fungus Magnaporthe oryzae, the causal agent of rice blast disease, contains the MGG_00245 gene, which encodes a putative PMO referred to as MoAA16. MoAA16 has been found to be highly expressed in planta during the early stages of fungal infection. The gene was optimized for heterologous expression in Pichia pastoris, and its oxidative cleavage activity on cellulose was characterized by analyzing soluble oligosaccharide products using highperformance anion exchange chromatography (HPAEC-PAD). The reaction catalyzed by MoAA16 requires 2 electrons from an electron donor, such as ascorbic acid, and aerobic conditions. It primarily produces Glc1 to Glc4 oligosaccharides, as well as oxidized cellobionic and cellotrionic acids. MoAA16 has been observed to enhance cellulase hydrolysis on phosphoric acid swollen cellulose (PASC) substrate, resulting in the production of more monosaccharide products. ConclusionsOur findings reveal the successful heterologous expression of MoAA16 in P. pastoris and its cellulose-active PMO properties. These results highlight the potential of MoAA16 as a promising candidate for applications in biofuel production and chemical synthesis.How to cite: Nguyen HM, Le LQ, Sella L, et al. Heterologous expression and characterization of a MoAA16 polysaccharide monooxygenase from the rice blast fungus Magnaporthe oryzae. Electron J Biotechnol 2023. https://doi.org/10.1016/j.ejbt.2023.06.002.

Effects and Stress-Relieving Mechanisms of Dark Tea Polysaccharide in Human HaCaT Keratinocytes and SZ95 Sebocytes.

A new skincare application scenario for dark tea, a unique and post-fermented tea popular in the health food industry, was developed in this paper. The effects of dark tea polysaccharide (DTP) on stress-induced skin problems and its mechanism of action were investigated by modeling cortisone-induced stress injury in human HaCaT keratinocytes and SZ95 sebaceous gland cells. The results showed a reduced cortisol conversion induced by cortisone under the action of DTP with a concentration of 200 μg/mL, probably by inhibiting the expression of the HSD11B1 enzyme. DTP was also able to suppress the cortisone-induced elevation of lipid levels in SZ95 sebocytes at this concentration. In addition, the composition and structure of DTP were verified by ultrafiltration, ultraviolet-visible spectrophotometry (UV-VIS), high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and infrared spectroscopy. In brief, DTP has a unique and significant stress-relieving effect, which provides new ideas for the development of new ingredients for the skin care industry.

Effect of environmental parameters on the content and sugar composition of sulfated polysaccharides in some tropical seagrass

Seagrasses are a paraphyletic group of marine angiosperms that evolved three to four times from land plants and returned to the sea. Halophila ovalis, Thalassia hemprichii and Enhalus acoroides (Hydrocharitaceae) are species that can occur in wide salinity ranges. Sulfated polysaccharides (SPs) comprise a complex group of macromolecules with many critical biological functions. We assume that SP may play a role in salt tolerance in seagrass. In this study, three seagrass species collected in both rainy and dry seasons from the fields were analyzed to determine the total SP contents and different functional groups of SP. Quantification of total SP was done by photometric assays. High-performance anion-exchange chromatography with Pulsed Electrochemical Detection (HPAEC) determined different functional groups of SPs. The results indicated higher total SP contents in seagrass are present in plants at higher salinities and environmental temperatures. The percent of functional groups of SPs are present in the following order: glucose &gt; galactose &gt; arabinose &gt; mannose &gt; rhamnose &gt; fucose. The order is not different between the two seasons.

Development of tandem cation exchange chromatography for high purity extracellular vesicle isolation: The effect of ligand steric availability

Purification of extracellular vesicles for research and therapeutic applications requires updated methodology to address the limitations of traditional ultracentrifugation and other size-based separation techniques. Their downfalls include induced extracellular vesicle aggregation, low yields, poor scalability and one-dimensionality of the separation process, as the size or sedimentation speed of extracellular vesicles is often the only selection criterion. Ion exchange chromatography is a promising alternative or supplementary method candidate, as it offers a different approach for extracellular vesicle separation, which is surface charge. For now, mostly anion exchange chromatography has been evaluated for extracellular vesicle purification, as it successfully relies on the strongly negative surface charge of extracellular vesicles. However, as extracellular vesicles are very complex in their structure, also cation exchange chromatography could be applicable, due to individual cationic domains on the extracellular vesicle surface. Here, we compare anion exchange chromatography to different types of cation exchange chromatography for the purification of platelet extracellular vesicle samples also containing plasma-derived impurities. We found that the choice of resin structure used for cation exchange chromatography is critical for binding platelet extracellular vesicles, as a conventional-type cation exchanger was found to only capture and elute less than 20% of extracellular vesicles. With the tentacle-type resin, it was possible to obtain comparable platelet extracellular vesicle yields (over 90%) with cation exchange chromatography compared to anion exchange chromatography, as well as superior purity, especially when it was combined to conventional cation exchange resin.

High performance anion exchange chromatography purification of probiotic bacterial extracellular vesicles enhances purity and anti-inflammatory efficacy.

Bacterial extracellular vesicles (BEVs), including outer membrane vesicles, have emerged as a promising new class of vaccines and therapeutics to treat cancer and inflammatory diseases, among other applications. However, clinical translation of BEVs is hindered by a current lack of scalable and efficient purification methods. Here, we address downstream BEV biomanufacturing limitations by developing a method for orthogonal size- and charge-based BEV enrichment using tangential flow filtration (TFF) in tandem with high performance anion exchange chromatography (HPAEC). The data show that size-based separation coisolated protein contaminants, whereas size-based TFF with charged-based HPAEC dramatically improved purity of BEVs produced by probiotic Gram-negative Escherichia coli and Gram-positive lactic acid bacteria (LAB). Escherichia coli BEV purity was quantified using established biochemical markers while improved LAB BEV purity was assessed via observed potentiation of anti-inflammatory bioactivity. Overall, this work establishes orthogonal TFF + HPAEC as a scalable and efficient method for BEV purification that holds promise for future large-scale biomanufacturing of therapeutic BEV products.

Non-targeted and suspect screening analysis using ion exchange chromatography-Orbitrap tandem mass spectrometry reveals polar and very mobile xenobiotics in Danish drinking water

Non-targeted and suspect screening analysis is gaining approval across the scientific and regulatory community to monitor the chemical status in the environment and thus environmental quality. These holistic screening analyses provides the means to perform suspect screening and go beyond to discover previously undescribed chemical pollutants in environmental samples. In a case study, we developed and optimized a high-resolution tandem mass spectrometry platform hyphenated with anion exchange chromatography to screen drinking water samples in Denmark. The optimized non-targeted screening method was able to detect anionic and polar compounds and was successfully applied to drinking water from two drinking water facilities. Following a data analysis pipeline optimization, anionic pesticide residues and other environmental contaminants were detected at confidence identification level 1 such as dimethachlor ESA, mecoprop, and dichlorprop in drinking water. In addition to these three substances, it was possible to detect another 1662 compounds, of which 97 were annotated at confidence identification level 2. More research is urgently needed to health risk prioritize the detected substances and to determine their concentrations.

Improved endotoxin removal using ecofriendly detergents for intensified plasmid capture.

Increasing plasmid demand for both production of viral and gene therapies as well as nucleic acid based vaccines has highlighted bottlenecks in production. One bottleneck is traditional bead-based chromatography as a capture step. To meet the needs of fast-growing markets, new production solutions are needed. These solutions must enable efficient capture of a diverse range of plasmid types and excellent clearance of bacterial host impurities, such as endotoxin. Enhanced endotoxin clearance during chromatographic purification has previously been demonstrated with detergents such as Triton™ X-100. However, degradation products of Triton™ X-100 are known to have a negative environmental impact, and more sustainable, environmentally benign alternatives have been identified. This work establishes an efficient, intensified plasmid capture using convective anion exchange (AEX) chromatography. The feasibility of the intensified capture approach was assessed with different membrane and a monolith AEX supports. Various detergents from different physico-chemical classes were evaluated with different AEX technologies. Purification efficiency evaluated endotoxin and host cell protein (HCP) clearance, plasmid yield, potential interference of the detergents with analytical in-process control assays, and overall process compatibility. This comprehensive screening approach provides valuable insights to intensified plasmid production.

Insights into product and process related challenges of lentiviral vector bioprocessing.

Lentiviral vectors (LVs) are used in advanced therapies to transduce recipient cells for long term gene expression for therapeutic benefit. The vector is commonly pseudotyped with alternative viral envelope proteins to improve tropism and is selected for enhanced functional titers. However, their impact on manufacturing and the success of individual bioprocessing unit operations is seldom demonstrated. To the best of our knowledge, this is the first study on the processability of different Lentiviral vector pseudotypes. In this work, we compared three envelope proteins commonly pseudotyped with LVs across manufacturing conditions such as temperature and pump flow and across steps common to downstream processing. We have shown impact of filter membrane chemistry on vector recoveries with differing envelopes during clarification and observed complete vector robustness in high shear manufacturing environments using ultra scale-down technologies. The impact of shear during membrane filtration in a tangential flow filtration-mimic showed the benefit of employing higher shear rates, than currently used in LV production, to increase vector recovery. Likewise, optimized anion exchange chromatography purification in monolith format was determined. The results contradict a common perception that lentiviral vectors are susceptible to shear or high salt concentration (up to 1.7 M). This highlights the prospects of improving LV recovery by evaluating manufacturing conditions that contribute to vector losses for specific production systems.

Protein-DNA Conjugates with a Discrete Number of Oligonucleotide Strands for Highly Reproducible Protein Quantification by the DNA Proximity Assay.

Protein-oligonucleotide conjugates are increasingly used as detection probes in biological applications such as proximity sensing and spatial biology. The preparation of high-quality conjugate probes as starting reagents is critical for achieving good and consistent performance, which we demonstrate via the DNA proximity assay (DPA) for the one-pot quantification of protein targets. We first established a complete conjugation and anion-exchange chromatography purification workflow to reproducibly obtain pure subpopulations of protein probes carrying a discrete number of oligonucleotide strands. A systematic study using the purified conjugate sub-populations confirmed that the order of conjugate (number of oligonucleotides per protein) and its purity (the absence of the unconjugated antibody) were important for ensuring optimal and reproducible assay performance. The streamlined workflow was then successfully used to conjugate a pair of universal DPA initiator oligonucleotides onto a wide range of binders including antibodies, nanobodies, and antigens which enabled the versatile detection of different types of proteins such as cytokines, total antibodies, and specific antibody isotypes. The good assay robustness (the inter-assay coefficient of variation lower than 5%) and linear calibration curve was achieved across all targets with just a single mix-and-incubate reaction step and a short reaction time of 30 min. We anticipate the streamlined protein-oligonucleotide probe preparation workflow developed in this work to have broad utility across applications leveraging the specificity of protein bio-recognition with the programmability of DNA hybridization.

Anti-Inflammatory and Anti-Oxidative Effects of Polysaccharides Extracted from Unripe Carica papaya L. Fruit.

This study evaluated the antioxidative and anti-inflammatory activities of polysaccharides extracted from unripe Carica papaya L. (papaya) fruit. Three papaya polysaccharide (PP) fractions, namely PP-1, PP-2, and PP-3, with molecular weights of 2252, 2448, and 3741 kDa, containing abundant xylose, galacturonic acid, and mannose constituents, respectively, were obtained using diethylaminoethyl-Sepharose™ anion exchange chromatography. The antioxidant capacity of the PPs, hydroxyl radical scavenging assay, ferrous ion-chelating assay, and reducing power assay revealed that the PP-3 fraction had the highest antioxidant activity, with an EC50 (the concentration for 50% of the maximal effect) of 0.96 mg/mL, EC50 of 0.10 mg/mL, and Abs700 nm of 1.581 for the hydroxyl radical scavenging assay, ferrous ion-chelating assay, and reducing power assay, respectively. In addition, PP-3 significantly decreased reactive oxygen species production by 45.3%, NF-κB activation by 32.0%, and tumor necrosis factor-alpha and interleukin-6 generation by 33.5% and 34.4%, respectively, in H2O2-induced human epidermal keratinocytes. PP-3 exerts potent antioxidative and anti-inflammatory effects; thus, it is a potential biofunctional ingredient in the cosmetic industry.

Physicochemical characterization and cosmetic application of kelp blanching water polysaccharides

Seaweeds account for half of global mariculture and have become a key player in bio-based industries. Seaweed process typically starts with hot water blanching that helps reduce postharvest quality deterioration but also generates large amounts of hydrothermal waste. This study aims to explore the feasibility of isolating water-soluble biopolymers from seaweed hydrothermal waste and their potential applications. Using Saccharina japonica (formerly Laminaria japonica) blanching water as example, 2.9 g/L of polymeric substances were efficiently isolated by ultrafiltration, implying biopolymer coproduction potential of ~5.8 kt from blanching wastewater of current kelp industry. Physicochemical characterizations revealed polysaccharidic nature of the biopolymers, with high contents of fucose, uronic acids and sulfate, showing distinct but also overlapping structural features with hot water-extracted kelp polysaccharides. The main fraction of the blanching water polymers after anion exchange chromatography was acidic polysaccharide, the major backbone residues of which were (1–4) linked mannopyranose, (1–4) linked gulopyranose and (1–2) linked fucopyranose while the branched residues were primarily 1,3,4-, 1,2,4- and 1,4,6-linked hexoses but also 1,3,4-fucopyranose. Furthermore, the polysaccharides were found to have a good compatibility in cosmetic creams with added cohesiveness and freshness, demonstrating the application potential of such natural biopolymers from currently underexplored seaweed blanching water.

Human serum albumin-bound selenium (Se-HSA) in serum and its correlation with other selenium species

IntroductionSelenium (Se) is a trace element with different toxicological and nutritional properties according to its chemical forms. Among the wide range of selenium species, human serum albumin-bound selenium (Se-HSA) has still uncertain composition in terms of organic or inorganic selenium species. This study aimed at investigating the relation between Se-HSA levels with total selenium and the specific organic and inorganic selenium species. MethodsWe determined levels of total selenium and selenium species in serum of participants enrolled in two populations of the Emilia-Romagna region, in Northern Italy. Anion exchange chromatography coupled with inductively coupled plasma dynamic reaction cell mass spectrometry was used as quantification method. Correlations between Se-HSA and the other selenium compounds were analyzed using linear regression and restricted cubic spline regression models, adjusted for potential confounders. ResultsThe first cohort comprised 50 participants (men/women: 26/24) with median (interquartile range, IQR) age 50 (55−62) years, while the second was composed of 104 participants (M/W: 50/54), median (IQR) age 48 (44−53) years. Median (IQR) levels of total selenium were 118.5 (109−136) µg/L and 116.5 (106−128) µg/L, respectively, while Se-HSA was 25.5 µg/L (16.2–51.5) and 1.1 (0.03–3.1) µg/L, respectively. In both populations, Se-HSA was positively associated with inorganic selenium species. Conversely, Se-HSA was inversely associated with organic selenium, especially with selenoprotein P-bound-Se (Se-SELENOP) and less strongly with selenomethionine-bound-Se (Se-Met), while the relation was null or even positive with other organic species. Evaluation of non-linear trends showed a substantially positive association with inorganic selenium, particularly selenite, until a concentration of 30 µg/L, above which a plateau was reached. The association with Se-SELENOP was inverse and strong until 100 µg/L, while it was almost null at higher levels. ConclusionsOur findings seem to indicate that Se-HSA incorporates more selenium when circulating levels of inorganic compounds are higher, thus supporting its mainly inorganic nature, particularly at high circulating levels of selenite.

Purification and characterization of a novel low-molecular-weight antimicrobial peptide produced by Lactiplantibacillus plantarum NMGL2

The present study aimed to purify and characterize a novel low-molecular-weight antimicrobial peptide (AMP) named as PNMGL2 produced by Lactiplantibacillus plantarum NMGL2. The AMP was effectively separated and purified by ethyl acetate extraction and DEAE-Sepharose anion exchange chromatography. Tricine-SDS-PAGE of the purified AMP showed a major protein band below 1.7 kDa, which was identified by MALDI-TOF MS to be a hexapeptide LNFLKK (761.95 Da), and structurally characterized to be combination of helixes and random coil by a PEP-FOLD 3 De novo approach. The antimicrobial activity of LNFLKK was confirmed by chemical synthesis of the peptide that showed clear inhibition (MIC 7.8 mg/mL) against both Gram-positive bacteria (Staphylococcus aureus and Listeria monocytogenes), and Gram-negative bacteria (Enterobacter sakazakii, Escherichia coli and Shigella flexneri). PNMGL2 was pH resistant (pH 2–9), heat stable (121 °C, 30 min), and protease sensitive. Treatment of UV rays, sodium chloride and organic solvents did not decrease the activity. Sequencing of the whole genome of L. plantarum NMGL2 revealed presence of a bacteriocin gene cluster with two putative bacteriocin genes (ORF4 and ORF5) that were not expressed, confirming the significance of PNMGL2 contributing the antimicrobial activity of the strain. This study demonstrated the low-molecular-weight AMP that was uncharacterized in the relevant available databases, suggesting its potential application as a novel natural food preservative.

Soluble expression and purification of recombinant bovine ferritin H-chain

Ferritin is a potential medicine delivery vehicle and vaccine platform, and its efficient expression is a prerequisite for widespread application. This study introduces a soluble expression strategy for recombinant bovine ferritin heavy chain (rFTH) in a prokaryotic system and an improved protein purification method. The amplified rFTH gene was ligated into the prokaryotic expression vector pET30a. The recombinant vectors with the N-terminal His-tag(N-His) or C-terminal His-tag(C-His) were translated and expressed separately. The results showed that the solubility of rFTH with C-His was significantly higher than that with N-His. The expression of rFTH with C-His was attempted at 37 °C and 16 °C, respectively. The results showed that the proportion of soluble protein expressed at 37 °C was more than 90%, higher than that expressed at 16 °C. Then rFTH with C-His was purified successfully using anion exchange chromatography, modified PEG precipitation, and dialysis. The rFTH protein was characterized using SDS-PAGE, Native-PAGE, Western blot, transmission electron microscopy, and dynamic light scattering. The results demonstrated that the purified rFTH protein self-assembled into ferritin nanoparticles with a regular shape and uniform size. This study sheds new light on the soluble expression of ferritin and provides a foundation for the construction of bovine ferritin nanoparticle production platforms.