Purification of Fusion Proteins by Affinity Chromatography on Glutathione Resin.

Fusion proteins that contain a glutathione S-transferase (GST) moiety can be purified to near homogeneity by affinity chromatography on glutathione-linked resins. Glutathione immobilized on a chromatography matrix, such as agarose or Sepharose, acts as a substrate for the GST moiety of fusion proteins. Contaminating proteins are washed away, and the bound GST fusion proteins are then readily displaced from the resin by elution with buffers containing free glutathione.

Rapid and Easy Enrichment Strategy for Naturally Acetylated N Termini Based on LysN Digestion and Amine-Reactive Resin Capture.

Protein N-terminal acetylation (Nα-acetylation) is one of the most common modifications in both eukaryotes and prokaryotes. Although studies have shown that Nα-acetylation plays important roles in protein assembly, stability, and location, the physiological role has not been fully elucidated. Therefore, a robust and large-scale analytical method is important for a better understanding of Nα-acetylation. Here, an enrichment strategy was presented based on LysN digestion and amine-reactive resin capture to study naturally acetylated protein N termini. Since LysN protease cleaves at the amino-terminus of the lysine residue, all resulting peptides except naturally acetylated N-terminal peptides contain free amino groups and can be removed by coupling with AminoLink Resin. Therefore, the naturally acetylated N-terminal peptides were left in solution and enriched for further liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The method was very simple and fast, which contained no additional chemical derivatization except protein reduction and alkylation necessarily needed in bottom-up proteomics. It could be used to study acetylated N termini from complex biological samples without bias toward different peptides with various physicochemical properties. The enrichment specificity was above 99% when it was applied in HeLa cell lysates. Neo-N termini generated by endogenous degradation could be directly distinguished without the use of stable-isotope labeling because no chemical derivatization was introduced in this method. Furthermore, this method was highly complementary to the traditional analytical methods for protein N termini based on trypsin only with ArgC-like activity. Therefore, the described method was beneficial to naturally acetylated protein N termini profiling.

Enhancing effect of macroporous adsorption resin on gamma-aminobutyric acid production by Enterococcus faecium in whole-cell biotransformation system.

Gamma-aminobutyric acid (GABA) biosynthesis depended to a great extent on the biotransformation characterization of glutamate decarboxylase (GAD) and process conditions. In this paper, the enhancing effect of D101 macroporous adsorption resin (MAR) on the GABA production was investigated based on the whole-cell biotransformation characterization of Enterococcus faecium and adsorption characteristics of D101 MAR. The results indicated that the optimal pH for reaction activity of whole-cell GAD and pure GAD was 4.4 and 5.0, respectively, and the pH range retained at least 50% of GAD activity was from 4.8 to 5.6 and 4.0-4.8, respectively. No substrate inhibition effect was observed on both pure GAD and whole-cell GAD, and the maximum activity could be obtained when the initial L-glutamic acid (L-Glu) concentration exceeded 57.6 mmol/L and 96.0 mmol/L, respectively. Besides, GABA could significantly inhibit the activity of whole-cell GAD rather than pure GAD. When the initial GABA concentration of the reaction solution remained 100 mmol/L, 33.51 ± 9.11% of the whole-cell GAD activity was inhibited. D101 MAR exhibited excellent properties in stabilizing the pH of the conversion reaction system, supplementing free L-Glu and removing excess GABA. Comparison of the biotransformation only in acetate buffer, the GABA production, with 50 g/100 mL of D101 MAR, was significantly increased by 138.71 ± 5.73%. D101 MAR with pre-adsorbed L-Glu could significantly enhance the production of GABA by gradual replenishment of free L-Glu, removing GABA and maintaining the pH of the reaction system, which would eventually make the GABA production more economical and eco-friendly.

Ex vivo investigation on internal tunnel approach/internal resin infiltration and external nanosilver-modified resin infiltration of proximal caries exceeding into dentin.

This ex vivo proof-of-concept study aimed to investigate the effect of nanosilver particles (AgNP) added to a conventional infiltrant resin (Icon) on external penetration into natural proximal enamel caries exceeding into dentin after internal tunnel preparation and internal infiltration. Carious lesions (ICDAS codes 2/3) of extracted human (pre-)molars revealing proximal caries radiographically exceeding into dentin (E2/D1 lesions) were preselected. Then, 48 of those specimens showing demineralized areas transcending the enamel-dentin border as assessed by means of near-infrared light transillumination (DIAGNOcam) were deproteinized (NaOCl, 5%). Using an internal tunnel approach, occlusal cavities central to the marginal ridge were prepared. Excavation of carious dentin, total etch procedure (H3PO4, 40%), and internal resin infiltration (FITC-labeled) followed, along with final restorations (flowable composite resin). Outer lesion surfaces were etched (HCl, 15%) prior to external infiltration (RITC-labeled). Group 1 (control; n = 24) used non-modified infiltrant, while an infiltrant/AgNP mixture (20 nm; 5.5 wt%) was used with experimental Group 2 (n = 24). Non-infiltrated pores of cut lesions were stained (Berberine), and specimens were analyzed using confocal laser scanning microscopy. Compared to the non-filled infiltrant, incorporation of AgNP had no effect on the resin's external penetration. Between the groups, no significant differences regarding internal or external infiltration could be detected, and non-infiltrated lesion areas did not differ significantly (p>0.109; t-test). The internal tunnel preparation in combination with both an internal resin infiltration and an additional external infiltration approach using a nanosilver-modified infiltrant resin leads to increased infiltrated lesion areas, thus occluding and adhesively stabilizing the porous volume of the demineralized enamel. While exerting antimicrobial effects by the nanosilver particles, this approach should have the potential as a viable treatment alternative for proximal lesions extending into dentin, thus avoiding the sacrifice of sound enamel, postponing the frequently inevitable restoration/re-restoration cycle of conventional proximal caries treatment, and improving dental health.

Biological Modification of Montan Resin from Lignite by Bacillus benzoevorans.

The montan resin (MR) is a solid waste produced during the industrial process of refined montan wax from lignite, and usually disposed by landfill and incineration, which easily cause environmental pollution and resource waste. Based its physicochemical properties, our study attempted to modify MR by Bacillus benzoevorans to achieve ecological utilization of MR. As results, the weight loss rate of MR, expressed as modification degree, was found to increase with the increase of B. benzoevorans-incubated time. The apparent oil-water partition coefficient (Kow), used to evaluate the improvement on hydrophilicity of MR, significantly increased (P < 0.01) after modification. IR analysis showed the functional groups of -OH and C=O in modified MR were more than those in MR. Meanwhile, comparison of the chemical changes between MR and modified MR by relatively quantitative analysis of gas chromatography-mass spectrometry (GC-MS) revealed that the content of some chemical components in the latter decreased, and the newly appeared chemical components all had more oxygen-containing functional groups. The bioactivity of the modified MR in agricultural application was evaluated regarding germination and seedling growth of maize seed preliminarly. Compared with the original MR-treated group, the modified MR showed an obvious effect on promoting the growth and germination of maize.

Microtensile bond strength, 4-point bending and nanoleakage of resin-dentin interfaces: Effects of two matrix metalloproteinase inhibitors.

Chronic degradation of hybrid layer collagen by matrix metalloproteinases (MMPs) jeopardizes resin-dentin interfacial integrity and limits the durability of dental restorations. The 4-point bending strength (BS) is a valid but uncommon method of testing the mechanical behavior of resin-dentin interfaces. The present study aims to analyze the influence of two matrix metalloproteinase inhibitors on microtensile bond strength (µTBS), BS and nanoleakage. A total of 48M were divided into three groups according to bonding procedure. Teeth were horizontally sectioned to produce a flat dentin surface. In the control group, etch-and-rinse Prime&Bond One (Dentsply) bonding was used; in the self-etch group, methacryloyloxydodecylpyridinium bromide (MDPB)-containing Clearfil SE Protect (Kuraray) was used; and in the benzalkonium chloride (BAC)-etch group, BAC-etchant (Bisco) was used. A Ceram.X-One (Dentsply) composite was built as three successive layers and was light-cured. Samples were sectioned to produce microrods that were randomly divided into two groups for analysis at baseline and after 6 months of water immersion (n = 32), plus one slab for nanoleakage analysis (n = 8) via scanning electron microscopy (SEM) and digital image analysis (Fiji). Data were analyzed using the Weibull distribution and a mixed-model ANOVA with a post hoc Tukey test. All groups showed deterioration of the initial bonds. The self-etch group had a worse baseline µTBS than the control but had the best BS after aging. BAC-etch did not improve bond stability of etch-and-rinse adhesive. The µTBS and BS test results after aging were moderately correlated. Mixed fractures prevailed with regard to µTBS, whereas adhesive fractures dominated with regard to BS. Nanoleakage was not eliminated in any group and increased after aging. MDPB self-etch resisted bond degradation better than etch-and-rinse adhesives, even after BAC-etching. Integrating BS in studies of µTBS and nanoleakage might provide more clinically relevant outcomes for predicting the performance of dental adhesives.

Novel High-throughput Approach for Purification of Infectious Virions.

Viruses are extensively studied as pathogens and exploited as molecular tools and therapeutic agents. Existing methods to purify viruses such as gradient ultracentrifugation or chromatography have limitations, for example demand for technical expertise or specialized equipment, high time consumption, and restricted capacity. Our laboratory explores mutations in oncolytic reovirus that could improve oncolytic activity, and makes routine use of numerous virus variants, genome reassortants, and reverse engineered mutants. Our research pace was limited by the lack of high-throughput virus purification methods that efficiently remove confounding cellular contaminants such as cytokines and proteases. To overcome this shortcoming, we evaluated a commercially available resin (Capto Core 700) that captures molecules smaller than 700 kDa. Capto. Core 700 chromatography produced virion purity and infectivity indistinguishable from CsCl density gradient ultracentrifugation as determined by electron microscopy, gel electrophoresis analysis and plaque titration. Capto Core 700 resin was then effectively adapted to a rapid in-slurry pull-out approach for high-throughput purification of reovirus and adenovirus. The in-slurry purification approach offered substantially increased virus purity over crude cell lysates, media, or high-spin preparations and would be especially useful for high-throughput virus screening applications where density gradient ultracentrifugation is not feasible.

Mechanistic understanding of fouling of protein A chromatography resin.

This paper aims to provide a thorough understanding of how fouling of Protein A resin takes place. Binding and mass transport properties of widely used agarose-based Protein A resin, MabSelect SuRe™, have been examined to understand the mechanism of resin fouling. There could be various factors that impact resin fouling. These include product/impurity build-up due to components in the feed material and ligand degradation due to the use of harsh buffers. To unravel their contributions, cycling studies were performed with and without product loading. The results presented in this paper provide a lucid understanding of the causative factors that limit Protein A chromatographic resin lifetime. The capacity fall for protein A resin at the end of 100th cycle due to use of feed material was found to be five times greater than that without using feed material. Compared to the fresh resin, the cycled resin samples shows 24% reduction in particle porosity and 51% reduction in pore mass transfer coefficient. Transmission electron microscopy (TEM) was used to qualitatively monitor accumulation of foulants on the cycled resin. Fouled resin sample contained a dense residue in the interior and exterior of resin particle both as a film at the bead surface and as granules. The surface activation energy increased five times in the case of fouled resin sample. The major event in fouling was identified as the non-specific adsorption of the feed material components on resin, signaling that pore diffusion is the rate limiting step. It is anticipated that these findings will assist in development of a more robust and economical downstream manufacturing process for monoclonal antibody purification.

Synthesis and thermal degradation studies of melamine formaldehyde resins.

Melamine formaldehyde (MF) resins have been synthesized at different reaction temperature and pH values. Different molar ratios of melamine and formaldehyde were used to synthesize the corresponding resins. The prepared resin samples were characterized by using molecular weight determination viscometry and thermogravimetric analysis (TGA). The maximum percentage of solid content (69.7%) was obtained at pH 8.5 and 75°C temperature. The molecular weight of MF resin was increased with an increase of melamine monomer concentration. The highest residual weight 14.125 wt.% was obtained with sample 10.

Identification of dengue-specific human antibody fragments using phage display.

High-affinity antibodies are valuable tools for dengue research. A method for the selection of dengue-specific, human antibody fragments using naïve repertoires displayed on M13 filamentous bacteriophage is described. Naïve repertoires are unbiased, thus enabling the identification of antibodies to dengue structural and nonstructural proteins from the same library. Dengue-specific clones are enriched by binding to an immobilized dengue antigen, followed by washing, elution, and amplification of phage for subsequent rounds of selection. Dengue virus has four antigenically related serotypes, and the serotype of the antigen can be kept constant or alternated during the selection process depending on whether serotype-specific or cross-reactive antibodies are required. After the selection process, clones are screened, and specific clones are identified by phage ELISA and Western blot.

Use of in situ solid-phase adsorption in microbial natural product fermentation development.

It has been half a century since investigators first began experimenting with adding ion exchange resins during the fermentation of microbial natural products. With the development of nonionic polymeric adsorbents in the 1970s, the application of in situ product adsorption in bioprocessing has grown slowly, but steadily. To date, in situ product adsorption strategies have been used in biotransformations, plant cell culture, the production of biofuels, and selected bulk chemicals, such as butanol and lactic acid, as well as in more traditional natural product fermentation within the pharmaceutical industry. Apart from the operational gains in efficiency from the integration of fermentation and primary recovery, the addition of adsorbents during fermentation has repeatedly demonstrated the capacity to significantly increase titers by sequestering the product and preventing or mitigating degradation, feedback inhibition and/or cytotoxic effects. Adoption of in situ product adsorption has been particularly valuable in the early stages of natural product-based drug discovery programs, where quickly and cost-effectively generating multigram quantities of a lead compound can be challenging when using a wild-type strain and fermentation conditions that have not been optimized. While much of the literature involving in situ adsorption describes its application early in the drug development process, this does not imply that the potential for scale-up is limited. To date, commercial-scale processes utilizing in situ product adsorption have reached batch sizes of at least 30,000 l. Here we present examples where in situ product adsorption has been used to improve product titers or alter the ratios among biosynthetically related natural products, examine some of the relevant variables to consider, and discuss the mechanisms by which in situ adsorption may impact the biosynthesis of microbial natural products.

Synthesis and swelling properties of ß-cyclodextrin-based superabsorbent resin with network structure.

A biodegradable, ß-cyclodextrin-based superabsorbent resin was synthesized by the inverse suspension method. The microstructure, chemical structure, and thermal performance of the resin were characterized by scanning electron microscopy, Fourier transform-infrared spectroscopy, and differential scanning calorimetry. The effects of the synthesis conditions (dosage of cross-linking agent, mass ratios of acrylic acid to acrylamide, mass ratios of ß-cyclodextrin to total monomer, neutralization degree, initiator dosage, and reaction time) were optimized to achieve a resin with a maximum swelling capacity. The water absorbency of the optimized resin in distilled water was 1544.76 g/g and that in 0.9 wt.% NaCl was 144.52 g/g. The resin, which is thermoplastic as well as pH-sensitive, had good salt resistance and underwent a maximum in swelling with time in CaCl(2) and AlCl(3) solutions. The fracture surface of the dry resin contained many pores. After swelling, the internal hydrogel showed a typical three-dimensional network structure. The biodegradation of the resin reached 71.2% after 18 days treatment at 30 °C with Lentinus edodes.

Synthesis, characterization and application of a new chelating resin for on-line separation, preconcentration and determination of Ag(I) by flame atomic absorption spectrometry.

A simple and reliable on-line separation/preconcentration procedure was developed for the determination of trace levels of Ag(I) by flame atomic absorption spectrometry. Poly[N-(3-methyl-1H-indol-1-yl)-2-methacrylamide-co-2-acrylamido-2-methyl-1-propane sulfonic acid-co-divinylbenzene] was synthesized and characterized as a new chelating resin for the first time. Ag(I) was sorbed on the chelating resin, from which it could be eluted with 3 mol L(-1) HCl and then introduced directly to the nebulizer-burner system for flame atomic absorption spectrometry. The parameters influential on the determination of Ag(I) ions such as the pH of the sample solution, amount of resin, eluent type, interfering ions and flow variables were studied. Under the optimum conditions, the calibration graph obtained was linear over the concentration range of 2-20 µg L(-1). The detection limit of the method (3σ) was 0.3 µg L(-1) while precision was 1.5% (n=25) at the level of 10 µg L(-1) Ag(I). The limit of quantification for the method, based on 20 σ, was 2.0 µg L(-1). The enrichment factor was found to be 65 while the optimized sample volume was 13.6 mL. The accuracy of the method was performed by analyzing certified reference materials. The developed method was applied successfully for the determination of silver in different water samples with satisfactory results.

Proteome of melamine urinary bladder stones and implication for stone formation.

Melamine can cause urinary stones related to nephropathy of the kidney and hyperplasia or carcinoma of the bladder, but the mechanism of stone formation is not well understood. In this study, male rats were administered melamine for thirteen weeks to establish melamine bladder stone models and the stones were analysed by Fourier transform infrared (FTIR) spectroscopy, powder X-ray diffraction (XRD), energy dispersive X-ray (EDX) spectroscopy, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) and western blot, respectively, for the composition and proteome, and to explore the implication of proteins for stone formation. The results showed bladder stones were composed of predominant melamine and a few amount of proteins. The proteins had a wide range of molecular weights and 1051 proteins were identified. Gene Ontology (GO) classification of the identified proteins showed most proteins were from injured cells, involved in various metabolic processes and had binding functions. Of the identified proteins, there were a few inflammatory proteins and urinary proteins. Physicochemical characteristics of the identified proteins showed that 67.1% proteins' isoelectric points (pI) value was below 7.0, 91.1% proteins' grand average of hydropathicity (GRAVY) scores were below 0 and nearly half of the proteins were stable. Our data indicated proteins might play an important role in melamine bladder stone formation.

Differential resin-dentin bonds created after caries removal with polymer burs.

The objective of this article was to investigate the effect of carbide and polymer burs caries removal methods on the bond strength of different adhesives to dentin. Resin restorations were performed in sound and caries-affected dentin, after using polymer or carbide burs and bonding with four different adhesive (Single bond, SB; Clearfil SE bond, SEB; FL-Bond II, FLB; and Fuji II-LC, FUJI). Microtensile bond strength (MTBS) was measured. Data were analyzed with ANOVA and Student-Newman-Keuls tests. Debonded surfaces were observed by scanning electron microscopy. Bonded interfaces were examined using light microscopy (Masson's trichrome staining). In sound dentin, MTBS was similar for SEB and SB, and higher than that of FLB and FUJI. Bond strength to carbide bur prepared dentin was similar for SB, SEB, and FLB; FUJI presented the lowest. SB applied on polymer bur excavated dentin presented similar values to those of the carbide bur group; MTBS attained by SEB, FLB, and FUJI decreased when bonding to dentin treated with polymer burs; FUJI yielded pretesting failures in all specimens. Polymer burs created a thick smear layer that was not infiltrated by tested self-etching agents. The bonding effectiveness of self-etching and glass-ionomer-like adhesives to dentin decreased when polymer burs were used.

A highly selective Hsp90 affinity chromatography resin with a cleavable linker.

Over 200 proteins have been identified that interact with the protein chaperone Hsp90, a recognized therapeutic target thought to participate in non-oncogene addiction in a variety of human cancers. However, defining Hsp90 clients is challenging because interactions between Hsp90 and its physiologically relevant targets involve low affinity binding and are thought to be transient. Using a chemo-proteomic strategy, we have developed a novel orthogonally cleavable Hsp90 affinity resin that allows purification of the native protein and is quite selective for Hsp90 over its immediate family members, GRP94 and TRAP 1. We show that the resin can be used under low stringency conditions for the rapid, unambiguous capture of native Hsp90 in complex with a native client. We also show that the choice of linker used to tether the ligand to the insoluble support can have a dramatic effect on the selectivity of the affinity media.

Resin-tooth interfacial morphology and sealing ability of one-step self-etch adhesives: microleakage and SEM study.

OBJECTIVES: To compare microleakage of three self-etch adhesives and to analyze enamel surface morphology and interfacial morphology of resin-enamel and resin-dentin interface under scanning electron microscope (SEM). EXPERIMENTAL DESIGN: Study was conducted in 65 extracted human premolars. Class V cavities were prepared in 45 teeth and assigned to three groups (n = 15) according to three self-etch adhesives (OptiBond All-in-One, iBond, and Adper Prompt L-Pop). After restoration, 10 samples from each group were used to assess microleakage at enamel and dentin margin. Five samples from each group were used for analysis of interfacial morphology at resin-enamel and resin-dentin interface under SEM. Remaining 20 teeth were used to prepare flat enamel buccal surfaces to analyze the difference in surface morphology after treatment with three adhesives (n = 5 each) and 36% phosphoric acid treatment (n = 5). PRINCIPAL OBSERVATIONS: At enamel margin, Prompt L-Pop depicted least leakage of all the three adhesives and also showed best interfacial adaptation under SEM. At dentin margin, OptiBond All-in-One showed significant less leakage than iBond and Prompt L-Pop. On flat enamel surface, phosphoric acid produced the most retentive etching pattern when compared with the three adhesives. CONCLUSION: Prompt L-Pop showed the best bonding effectiveness in enamel, whereas OptiBond All-in-One performed significantly better in dentin.

Composting of waste paint sludge containing melamine resin as affected by nutrients and gypsum addition and microbial inoculation.

Melamine formaldehyde resins have hard and durable properties and are found in many products, including automobile paints. These resins contain high concentrations of nitrogen and, if properly composted, can yield valuable products. We evaluated the effects of starter compost, nutrients, gypsum and microbial inoculation on composting of paint sludge containing melamine resin. A bench-scale composting experiment was conducted at 55 °C for 91 days and then at 30 °C for an additional 56 days. After 91 days, the composts were inoculated with a mixed population of melamine-degrading microorganisms. Melamine resin degradation after the entire 147 days of composting varied between 73 and 95% for the treatments with inoculation of microorganisms compared to 55-74% for the treatments without inoculation. Degradation was also enhanced by nutrients and gypsum additions. Our results infer that large scale composting of melamine resins in paint sludge is possible.

Transplacental transfer of melamine.

OBJECTIVE: To characterize transplacental transfer of melamine and related mechanisms as well as toxicity using human placental perfusion and cultured cells. METHODS: Transfer and toxicity were analyzed in 4-h perfusions with 10 µM or 1 mM melamine, or 10 µM melamine with 10 nM cyanuric acid (CYA). Efflux transporters were studied in accumulation assay and toxicity in BeWo cells by MTT assay. RESULTS: Of added melamine 34-45% was transferred to fetal circulation and CYA made no difference. Histology, hCG production, and PLAP activity indicated functionality of placental tissue with no grave toxicity. Highest concentration of melamine used (2 mM) with CYA and long treatment time decreased viability of BeWo cells. Inhibitors of ABCB1, ABCG2, ABCC2 did not affect the accumulation of melamine in cells. CONCLUSION: Melamine goes through human term placenta with no contribution of efflux transporters. Toxicity of melamine is low in placental tissue and BeWo cells.

The effect of exogenous melamine on rat hippocampal neurons.

Rat hippocampal neurons in culture medium were exposed to different concentrations of melamine. By examining the morphology of cells detected with acridine orange staining, different changes of fluorescences of Ca²âº observed with Fluo3, and caspase-3 activity assayed with optical density by enzyme linked immunosorbent assay kit, we found the effect of melamine on hippocampal neurons. Pathologic changes happened in hippocampal neurons, and there seemed to be insoluble metabolites in most of the cells with 312 µg/mL melamine stimulated for 12 hours. Then, we tested the Ca²âº fluorescences of the cells above. The free intracellular Ca²âº concentrations were measured using the fluorescent dye Fluo3. The average fluorescence of Ca²âº in hippocampal neurons stimulated by 312 µg/mL melamine (55.43 ± 3.54) was higher than the normal ones (6.94 ± 0.14). Besides, caspase-3 activity of hippocampal neurons after being challenged with melamine was higher than that of normal ones in the mass. From the above conclusions, melamine did induce damnification to hippocampal neurons, probably in the way of inducing cells to undergo apoptotic processes and disrupting the homeostasis of Ca²âº. Our experimental results disproved some viewpoints that not melamine alone, but melamine and cyanuric acid in combination could cause damage in toxicological studies and provided compelling evidence that low levels of melamine exposure may also represent a health risk to nerves, in opposition to the idea that damage of melamine and cyanuric acid in combination were limited to the kidneys.