Two-Step Purification of L-Asparaginase from Acrylaway® L

Abstract L-Asparaginase (L-ASNase) is a biopharmaceutical used for acute lymphoblastic leukaemia (ALL) treatment, dramatically increasing the patients' chance of cure. However, its production and distribution in developing countries were disrupted because of its low profitability, which caused great concern among patients. This study evaluates the feasibility of combining fractional precipitation and aqueous two-phase systems (ATPS) to purify L-ASNase from a low-grade product, commercially known as Acrylaway® L. The ATPS purification results were not particularly expressive compared to the two-step purification process composed of ethanol precipitation and gel filtration, which was able to recover the target molecule with a purification factor over 5 fold. Thus, we studied a purification process capable of manufacturing pharmaceutical grade L-ASNase from a commercially available low-grade raw material; however, improvements regarding its throughput must be achieved, and high purity is the first step to apply it as a new biopharmaceutical product. The proposed process could pose as a short-time solution to mitigate its shortage while a cost-effective production plant is being developed.

Step gradient alcohol precipitation for the purification of low molecular weight fucoidan from Sargassum siliquastrum and its UVB protective effects.

Ultraviolet B (UVB) can induce oxidative damage to outermost layers of skin causing suntans, sunburns, and, in severe cases, blisters leading to photoaging. Low molecular weight (MW) fucoidan is renowned for possessing enhanced antioxidant activities. The present study discloses the use of step gradient ethanol precipitation in refining fucoidan fractions (SSQC1-SSQC4) from Sargassum siliquastrum and evaluation of their UVB-protective effects in human HaCaT keratinocytes. Among the fractions, SSQC4 indicated the best bioactive effects. 1H NMR, FTIR, monosaccharide composition by HPAEC-PAD analysis, MW estimation by agarose gel electrophoresis were used to characterize the fractions. SSQC4 was comprising of fucoidan, with an estimated MW distribution of 8-25 kDa. Exposure of UVB increased intracellular ROS, DNA damage, loss of mitochondrial membrane potential, apoptotic body formation causing cell death through the mitochondria-mediated apoptosis pathway. SSQC4 treatment could dose-dependently attenuate the ROS levels and suppress mitochondria-mediated apoptosis in UVB exposed keratinocytes. SSQC4 treatment enhanced cellular antioxidant defense by increasing Nrf2 mediated HO-1 generation, which was identified as the cause of observed bioactivities. The safety and stability of SSQC4 could be further evaluated to promote its use as a bioactive natural ingredient in UV-protective cosmetics.

Co-precipitation with CaCO3 to remove heavy metals and significantly reduce the moisture content of filter residue.

The co-precipitation of Fe2+, Cu2+, Zn2+, Cd2+ and Ni2+ were investigated by a mechanochemical processing with CaCO3. The results showed that the synergies of the metal ions led to efficient co-precipitation. The precipitation of Fe2+, Cu2+ and Cd2+ are over 99% and that of Zn2+ and Ni2+ about 98.4% and 93.8%. A significant advantage of the process is that the moisture content of filter residue is much lower (less than 50%) than that using the lime neutralization (more than 80%), offering a potential solution to the sludge problem in wastewater treatment. A further advantage is the neutral pH (about 7.5) obtained by using CaCO3 rather than the highly alkaline pH (about 11) obtained using lime (Ca(OH)2) neutralization method.

Urinary extracellular vesicles as a source of biomarkers reflecting renal cellular biology in human disease.

For more than a decade, extracellular vesicles (EVs) have been the focus of extensive research efforts attempting to uncover their biological function in health and disease. Likewise, numerous studies have investigated them as a source of potential biomarkers to complement or replace the routine diagnostic procedures. Urinary extracellular vesicles take a distinct place among these studies, as they hold the promise to reflect changes in the cellular biology of the nephron and can be isolated without any invasive procedure. However, their potential has been insufficiently exploited since both their biological function and their use for diagnostic purposes in human disease have only gained increasing attention in the last years. This review aims to give an overview of the present knowledge about urinary extracellular vesicles with a special focus on novel nomenclature recommendations, current techniques for urinary EV separation and potential biomarkers that have emerged from the analysis of urinary EVs.

Comparison of Plasma Exosomes by Differential Ultracentrifugation and Solvent Precipitation Methods.

BACKGROUND: Emerging evidence has identified that exosomes play a pivotal role in intercellular signal transmission. However, the standardized purification techniques to isolate high quality exosomes are still deficient at present. This study was to evaluate reproducibility and efficiency of differential ultracentrifugation and solvent precipitation-based kits by isolating plasma-derived exosomes from oral lichen planus patients. METHODS: Morphology, exosomal biomarkers, particle size distribution, proteomic components, and protein yield of isolated exosomes were evaluated by transmission electron microscope, western blot, laser diffraction instrument, Coomassie staining, and BCA protein assay kit, respectively. RESULTS: TEM displayed representative cup-shaped morphology of exosomes and western blot identified exosomal biomarkers CD9 and CD63. The size distribution showed that particles by differential ultracentrifugation were mainly from 26.15 nm to 166.5 nm, while some of the particles obtained by solvent precipitation kits were larger than 1,000 nm. In addition, exosomes isolated by solvent precipitation kits showed a significantly higher amount of protein yield due to plasma albumin contamination. CONCLUSIONS: Both differential ultracentrifugation and precipitation based kits could successfully isolate plasma exosomes, and exosomes by differential ultracentrifugation were purer and more appropriate for further proteomic analysis.

Effects of Fe(III)-fulvic acid on Cu removal via adsorption versus coprecipitation.

This study compared the sorption and extractability of Cu following adsorption (SOR) and coprecipitation(CPT). The effect of solution pH, Fe: organic carbon (OC) ratios and fulvic acid (FA) on the combined removal of Cu was investigated in the batch tests using Fe(III) precipitates as a sorbent. Transmission electron microscope (TEM) images demonstrated that the coexisting FA reduced the particle size of ferrihydrites as expected. Generally, more Cu was eliminated in coprecipitation compared with adsorption and the dissolved Cu left in solutions decreased as the pH increased, most of dissolved Cu was trapped at pH 6 and above. Meanwhile, the inhibition or promotion of Cu removal really depended on the different Fe: OC ratios. The addition of FA led to a further decrease of Cu concentrations in CPT systems with Fe/OC ratio of 1:3, however, Cu removal was hindered in the presence of FA in SOR systems. In the case of extraction experiments, the addition of l-malic acid (MA), oxalic acid (OA) and citric acid (CA) resulted in lower extractability of coprecipitated Cu than adsorption samples. The gaps in extractions were seemed to be a consequence of tight Cu binding in CPT products, and the more feasible desorption of Cu from the surface of SOR samples. Based on the results of Cu adsorption and coprecipitation, coprecipitation of Cu with ferrihydrites was the more effective Cu sequestration mechanism in the removal of Cu. These results are helpful to understand the complicated interactions among Fe(III), FA and Cu (II) in the natural environment.

Sample preparation for large-scale bioanalytical studies based on liquid chromatographic techniques.

Quality of the analytical data obtained for large-scale and long term bioanalytical studies based on liquid chromatography depends on a number of experimental factors including the choice of sample preparation method. This review discusses this tedious part of bioanalytical studies, applied to large-scale samples and using liquid chromatography coupled with different detector types as core analytical technique. The main sample preparation methods included in this paper are protein precipitation, liquid-liquid extraction, solid-phase extraction, derivatization and their versions. They are discussed by analytical performances, fields of applications, advantages and disadvantages. The cited literature covers mainly the analytical achievements during the last decade, although several previous papers became more valuable in time and they are included in this review.

Isolation and identification of antifungal peptides from Bacillus amyloliquefaciens W10.

Antifungal metabolites produced by Bacillus sp. W10, which was previously isolated from the tomato rhizosphere, were investigated. Strain W10 was identified as Bacillus amyloliquefaciens by analysis of its 16S rDNA and gyrB gene partial sequences. PCR analysis showed the presence of fenB, sfp, and ituD genes, coding for fengycin, surfactin, and iturin, respectively. A novel small antifungal peptide, designated 5240, produced by this strain was isolated by ammonium sulfate precipitation and Superdex 200 gel filtration chromatography. The 5240 peptide was stable at 100 °C for 20 min and remained active throughout a wide pH range (4-10). The antagonistic activity was not affected by protease K and trypsin. The purified 5240 peptide exhibited a broad inhibitory spectrum against various plant pathogenic fungi and was identified as iturin A (C14-C16). Moreover, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry indicated the presence of fengycin A (C14-C15), fengycin B (C16-C17), and surfactin (C13-C16) isoforms in supernatants from strain W10. These results suggest that B. amyloliquefaciens W10 has significant potential as a biocontrol agent.

Rapid quantification of multi-components in alcohol precipitation liquid of Codonopsis Radix using near infrared spectroscopy (NIRS).

A near infrared spectroscopy (NIRS) approach was established for quality control of the alcohol precipitation liquid in the manufacture of Codonopsis Radix. By applying NIRS with multivariate analysis, it was possible to build variation into the calibration sample set, and the Plackett-Burman design, Box-Behnken design, and a concentrating-diluting method were used to obtain the sample set covered with sufficient fluctuation of process parameters and extended concentration information. NIR data were calibrated to predict the four quality indicators using partial least squares regression (PLSR). In the four calibration models, the root mean squares errors of prediction (RMSEPs) were 1.22 µg/ml, 10.5 µg/ml, 1.43 µg/ml, and 0.433% for lobetyolin, total flavonoids, pigments, and total solid contents, respectively. The results indicated that multi-components quantification of the alcohol precipitation liquid of Codonopsis Radix could be achieved with an NIRS-based method, which offers a useful tool for real-time release testing (RTRT) of intermediates in the manufacture of Codonopsis Radix.

A reformative shear precipitation procedure for the fabrication of vancomycin-loaded poly(lactide-co-glycolide) microspheres.

This study reports the encapsulation of vancomycin, as a model hydrophilic drug, into poly(lactide-co-glycolide) microspheres using a novel reformative shear precipitation procedure. In contrast to the external aqueous phase used in the conventional microencapsulation technique based on emulsion solvent evaporation/extraction, the reformative shear precipitation procedure explored in this study uses a shear medium composed of glycerol as the viscous medium and ethanol as polymer antisolvent, which is relatively immiscible with the hydrophilic drug. This limits drug diffusion and leads to rapid microsphere solidification, which allows a large proportion of the hydrophilic drug to be encapsulated within the microspheres. The influence of various processing parameters, including polymer concentration, volume ratio of ethanol to glycerol in the shear medium, volume of aqueous drug solution, initial drug loading, and injecting rate of the drug-polymer emulsion, on the encapsulation efficiency and characteristics of resulting microspheres were investigated. The morphology and release characteristics, as well as mechanical, in vitro and in vivo behaviour of vancomycin-loaded poly(lactide-co-glycolide) microspheres prepared using the novel procedure were also investigated. The results demonstrated that the reformative shear precipitation procedure could achieve the loading of hydrophilic drugs into poly(lactide-co-glycolide) microspheres with high encapsulation efficiency, and the success of the procedure was largely influenced by the volume ratio of ethanol to glycerol in the shear medium. Vancomycin-loaded poly(lactide-co-glycolide) microspheres prepared using this procedure demonstrated favourable mechanical characteristics, antibacterial activity, and in vivo degradation behaviour which suggested their suitability for use as a sustained delivery system.

Facile synthesis of a ratiometric oxygen nanosensor for cellular imaging.

A new type of cell-penetrating ratiometric fluorescence oxygen sensing nanoparticle was prepared through a facile co-precipitation method. Amphiphilic polymer poly (styrene-co-maleic anhydride) (PSMA) was firstly cooperated with polystyrene (PS) to envelop the highly photostable phosphorescent oxygen indicator, platinum(II)-tetrakis(pentafluorophenyl)porphyrin (PtTFPP, emission at 648nm), and the reference fluorophore, poly(9, 9-dioctylfluorene) (PFO, emission at 440nm ), via hydrophobic interaction in aqueous solution. To improve the sensor biocompatibility, the biomacromolecule poly-l-lysine (PLL) was selected to act as a shell via electrostatic forces. The as-prepared PtTFPP doped core-shell nanoparticles (called PPMA/PLL NPs) exhibited an excellent ratiometric luminescence response to O2 content with high quenching efficiency and full reversibility in the oxygen sensing. More importantly, these oxygen nanosensors passed across the cell membrane after co-incubation without external force. Labeled cells exhibited high brightness in the matching blue and red channels of a digital camera. And most nanosensors were found locating in cytoplasm rather than being trapped in endosomes.

A convenient purification method for silver nanoclusters and its applications in fluorescent pH sensors for bacterial monitoring.

Herein, we report a convenient approach to purify water-soluble dihydrolipoic acid (DHLA)-capped Ag nanoclusters (Ag NCs) by pH-induced precipitation under acidic conditions. The fluorescence of Ag NCs could be completely recovered by re-dispersing the precipitate into a basic solution using DHLA and NaBH4 as stabilizing ligands and etching reagent. DHLA-Ag NCs-doped agarose hydrogels have been prepared to monitor pH with a wide range from 8.0 to 4.0. When pH decreased, the fluorescence of the hydrogels under a UV lamp decreased and completely disappeared after pH 5. The DHLA-Ag NCs-doped agarose hydrogels biosensor showed low cytotoxicity and long stability. Accordingly, a fluorescent pH sensor for bacterial monitoring has been employed based on the "OFF-ON" signal switch of the Ag NCs-agarose hydrogel.

A sensitive impedimetric platform biosensing protein: Insoluble precipitates based on the biocatalysis of manganese(III) meso-tetrakis (4-N-methylpyridiniumyl)-porphyrinin in HCR-assisted dsDNA.

In this study, a sensitive biosensing interface for protein was reported based on nonconductive insoluble precipitates (IPs) by the biocatalysis of manganese(III) meso-tetrakis (4-N-methylpyridiniumyl)-porphyrin (MnTMPyP), which was intercalated into formed double-strand DNA (dsDNA) scaffold triggered by hybridization chain reaction (HCR). In the proposed impedimetric aptasensor, carcinoembryonic antigen (CEA) and its aptamer were used as testing model. PtPd nanowires (PtPdNWs) with large surface area and superior conductivity were employed as nanocarriers to greatly immobilize biomolecules (e.g. CEA aptamers). Then, two DNA hairpins H1 and H2 were introduced to trigger HCR with the assistance of DNA initiator, resulting in the formation of a long dsDNA scaffold. Meanwhile, mimicking enzyme MnTMPyP molecules were embedded into the resultant dsDNA, in situ generating the complex MnTMPyP-dsDNA with peroxidase-like activity. Under the biocatalysis of MnTMPyP-dsDNA, 3,3-diaminobenzidine (DAB) was oxidized to form nonconductive IPs. As a result, the electron transfer between electrode interface and redox probe was vastly hindered, leading to the significant amplification of electrochemical impedimetric signal. So, greatly improved analytical performances of the proposed aptasensor were achieved with a detection limit as low as 0.030pgmL(-1). And the successful assay of CEA in human serum samples enabled the developed biosensing platform to have promising potential in bioanalysis.

Role of peach proteins in juice precipitation induced by high pressure CO2.

To better understand the role of peach proteins in juice precipitation induced by high pressure CO2 (HPCD), proteins extracted from peach juice were subjected to HPCD and heat, and changes in particle size distribution (PSD) and structure were investigated. PSD analysis showed aggregations of proteins were both induced by HPCD and heat, but HPCD induced a stronger aggregation. The endotherm of HPCD- and heat-treated proteins moved to lower temperature, indicating that higher-order structures were altered after treatments. Furthermore, proteins related to HPCD- and heat-induced precipitation were analyzed by proteomics and bioinformatics. It was found that proteins with low content of α-helix and hydrogen bonds were more inclined to precipitate under HPCD, and HPCD precipitated proteins with more compact structures than heat, which might cause the stronger aggregation of proteins by HPCD. In conclusion, HPCD could induce the aggregation of peach proteins by destroying higher-order structures, which contributes to juice precipitation.

Selective Precipitation of Proteins.

Selective precipitation of proteins can be used as a bulk method to recover the majority of proteins from a crude lysate, as a selective method to fractionate a subset of proteins from a protein solution, or as a very specific method to recover a single protein of interest from a purification step. This unit describes a number of methods suitable for selective precipitation. In each of the protocols that are outlined, the physical or chemical basis of the precipitation process, the parameters that can be varied for optimization, and the basic steps for developing an optimized precipitation are described.

Magnetic nanoparticles adapted for specific biomedical applications.

Magnetic nanoparticles (MNPs) are used in different biomedical applications, whereby each application requires specific particle properties. To fulfill these requirements, particle properties have to be optimized by means of variation of crystal structure, particle size, and size distribution. To this aim, improved aqueous precipitation procedures for magnetic iron oxide nanoparticle synthesis were developed. One procedure focused on the cyclic growth of MNPs without nucleation of new particle cores during precipitation. The second novel particle type are magnetic multicore nanoparticles, which consist of single cores of approximately 10 nm forming dense clusters in the size range from 40 to 80 nm. Their highest potential features these multicore particles in hyperthermia application. In our in vivo experiments, therapeutically suitable temperatures were reached after 20 s of heating for a particle concentration in the tumor of 1% and field parameters of H=24 kA/m and f=410 kHz. This review on our recent investigations for particle optimization demonstrates that tuning magnetic properties of MNPs can be obtained by the alteration of their structure, size, and size distribution. This can be realized by means of control of particle size during synthesis or subsequent size-dependent fractionation. The here-developed particles show high potential for biomedical applications.

Comparative studies of two methods for miRNA isolation from milk whey.

MicroRNAs (miRNAs) from milk whey have been considered for their potential as noninvasive biomarkers for milk quality control and disease diagnosis. However, standard protocols for miRNA isolation and quantification from milk whey are not well established. The objective of this study was to compare two methods for the isolation of miRNAs from milk whey. These two methods were modified phenol-based technique (Trizol LS(®) followed by phenol precipitation, the TP method) and combined phenol and column-based approach (Trizol LS(®) followed by cleanup using the miRNeasy kit, the TM method). Yield and quality of RNA were rigorously measured using a NanoDrop ND-1000 spectrophotometer and then the distribution of RNA was precisely detected in a Bioanalyzer 2100 instrument by microchip gel electrophoresis. Several endogenous miRNAs (bta-miR-141, bta-miR-146a, bta-miR-148a, bta-miR-200c, bta-miR-362, and bta-miR-375) and an exogenous spike-in synthetic control miRNA (cel-miR-39) were quantified by real-time polymerase chain reaction (PCR) to examine the apparent recovery efficiency of milk whey miRNAs. Both methods could successfully isolate sufficient small RNA (<200 nt) from milk whey, and their yields were quite similar. However, the quantification results show that the total miRNA recovery efficiency by the TM method is superior to that by the TP method. The TM method performed better than the TP for recovery of milk whey miRNA due to its consistency and good repeatability in endogenous and spike-in miRNA recovery. Additionally, quantitative recovery analysis of a spike-in miRNA may be more accurate to reflect the milk whey miRNA recovery efficiency than using traditional RNA quality analysis instruments (NanoDrop or Bioanalyzer 2100).

Application and reactivation of magnetic nanoparticles in Microcystis aeruginosa harvesting.

This study developed a magnetic nanoparticles (MNPs) harvesting and reactivation technique for rapid cyanobacteria Microcystis aeruginosa separation. The harvesting of raw MNPs achieved high efficiency of 99.6% with the MNPs dosage of 0.58g MNPs/g dry-biomass, but gradually decreased to 59.1% when directly reused 5 times. With extra ultrasonic chloroform:methanol solvent treatment, the MNPs can be effectively reactivated for M. aeruginosa harvesting with 60% efficiency after 5 times reactivation and the separation efficiency kept above 93% with 0.20g MNPs/g dry-biomass dosage. The cyanobacteria-MNPs complex can be effectively disrupted by ultrasonic chloroform:methanol solvent treatment and the zeta potential was recovered for MNPs electrostatic attraction. The MNPs adsorption followed the Langmuir isotherm, and the maximum adsorption capacity and Langmuir constant was 3.74g dry-biomass/g and 311.64L/g respectively. This MNPs reactivation technique can achieve low energy separation and reduce MNPs consumption by 67%, providing potential engineering implementation for cyanobacterial biomass harvesting.

Depletion of RuBisCO protein using the protamine sulfate precipitation method.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a major high-abundant protein (HAP) in the plant leaves which hinders analysis of low-abundant proteins (LAP). In this chapter, we describe a highly simple RuBisCO depletion method using protamine sulfate (PS). Addition of 0.1 % PS is sufficient to precipitate the RuBisCO from the leaf extracts of diverse plants including monocots and dicots. Our results of SDS-PAGE, Western blotting, and two-dimensional gel electrophoresis showed that both large and small subunits of RuBisCO were precipitated in the pellet fractions, while LAPs were enriched in the supernatant fraction after PS precipitation.

A modified precipitation method to isolate urinary exosomes.

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.