Alternative Purification Method Research Articles

A facile TiO2/PVDF composite membrane synthesis and their application in water purification

In this work, we have demonstrated a facile wet chemical method to synthesise TiO2/PVDF composite membranes as alternative water purification method to traditional polymer-based membrane. For the first time, hydrothermally grown TiO2 nanofibers under alkali conditions were successfully inserted into PVDF membranes matrix. The structure, permeability and anti-fouling performance of as-prepared PVDF/TiO2 composite membranes were studied systematically. The TiO2/PVDF composite membranes prepared in this work promise great potential uses in water purification applications as microfiltration membranes due to its excellent physical/chemical resistance, anti-fouling and mechanical properties.

Dictyonema black shale and Triassic sandstones as potential sources of uranium

Abstract The main objective of the present study was an assessment of the possibility of uranium recovery from domestic resources in Poland. In the first stage uranium was leached from the ground uranium ore by using acidic (sulfuric acid or hydrochloric acid) or alkaline (carbonate) solutions. The leaching efficiencies of uranium were dependent on the type of ore and it reached 81% for Dictyonemic shales and almost 100% for sandstones. The novel leaching routes, with the application of the helical membrane contactor equipped with rotating part were tested. The obtained postleaching solutions were concentrated and purified using solvent extraction or ion exchange chromatography. New methods of solvent extraction, as well as hybrid processes for separation and purification of the product, were studied. Extraction with the use of membrane capillary contactors that has many advantages above conventional methods was also proposed as an alternative purification method. The final product U3O8 could be obtained by the precipitation of ‘yellow cake’, followed by calcination step. The results of precipitation of ammonium diuranate and uranium peroxide from diluted uranium solution were presented

Purification of proteins from solutions containing residual host cell proteins via preparative crystallization.

To investigate quantitatively and reproducibly a scalable, preparative crystallization method in novel stirred tanks using three different protein solutions containing residual microbial host cell proteins (HCP). Lysozyme from solutions being spiked with up to 15% host cell proteins (HCP) (corresponding to 176,500 ppm) was crystallized within a 2.4-4.6 h at 93.7% yield using NaCl and glycerol. Lipase was crystallized under comparable conditions using NaCl and a mixture of two polyethylene glycols (PEG). Enhanced green fluorescent protein (eGFP) was overexpressed in E. coli yielding a solution containing 23% target protein. Residual HCP content after pre-treatment was 7-16%. eGFP was crystallized from these solutions within 1.75-4 h at 88.7% step yield using ethanol and the same mixture of two PEG as in the case of lipase. HCP contained in the solvent channels of the protein crystals could be removed by diffusive washing yielding final purities at or above 99%. Preparative crystallization can be carried out with fast kinetics and high yields from solutions containing residual impurities and may represent an attractive alternative purification method compared to preparative chromatography, especially at large production scales.

An alternative purification method for human serum paraoxonase 1 and its interactions with anabolic compounds

In this study, an alternative purification method for human paraoxonase 1 (hPON1) enzyme was developed using two-step procedures, namely, ammonium sulfate precipitation and Sepharose-4B-l-tyrosine-3-aminophenantrene hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent MW of 43 kDa. The enzyme was purified 219-fold with a final specific activity of 4 408 400 U/mg and a yield of 10%. Furthermore, we examined the in vitro effects of some anabolic compounds, such as zeranol, 17 β-estradiol, diethylstilbestrol, oxytocin, and trenbolone on the enzyme activity to understand the better inhibitory properties of these molecules. The five anabolic compounds dose dependently decreased the activity of hPON1 with inhibition constants in the millimolar–micromolar range. The results show that these compounds exhibit inhibitory effects on hPON1 at low concentrations with IC50 values ranging from 0.064 to 16.900 µM.

Bentonite, temperature and pH effects on purification indexes of raw sugar beet juice to production of inverted liquid sugar

The present study deals with modeling of the effects of processing temperature (30–95°C), concentration of Bentonite (Bent) (1–5g/L) and pH (3.5–6) on color, turbidity, purity, ash and invert sugar content of purified raw sugar-beet juice. The used methodology was the face central composite design of response surface to optimize the key parameters (temperature of processing, concentration of Bent and pH) of the process. The main goal was to focus on the possibility to produce invert sugar directly from raw sugar-beet juice in conjunction with an alternative purification method and also comparison with the common conventional lime processing in the industry. In this way, second-order polynomial models were developed for dependent responses using least-square fit of regression analysis. The results of analysis of variance indicated that all three investigated independent parameters have significant influence on purity indexes of raw beet-juice. The optimum condition was a constant reaction time (30min) at temperature of 75°C, pH4.47 and Bent concentration of 1.7g/L which were determined on minimization of color, turbidity and ash content and maximization of invert sugar and purity. At this optimum point: color, turbidity, purity, ash and invert sugar content were found to be 1664 ICU420, 6.3 NTU, 93.9%, 0.55% and 1.6%, respectively. Also the clarified juice quality obtained by Bent method was improved greatly when compared with conventional limed–carbonated as the color and turbidity in clarified juice were removed by the rate of 35.55% and 76.09%, respectively.

Alternative purification method for recombinant measles viral nucleoprotein expressed in insect cells by ion-exchange chromatography

Recombinant measles virus nucleoproteins (rMeV N) and fusion (F) proteins were characterized as major antigenic proteins expressed in insect cells mediated by recombinant baculoviruses (rBVs). Band intensities were analyzed by Western blotting to recognize IgG and IgM antibodies against the rMeV N and F proteins in human sera and cerebrospinal fluids (CSFs) from patients with measles infections. Positive results from the blots using the rMeV N were consistent with the results of enzyme-linked immunosorbent assays (ELISAs) in which whole viral proteins were used as antigens. Human sera and CSFs reacted more strongly with the rMeV N than with the rMeV F proteins prepared in an identical expression system. For efficient and reliable purification, ion-exchange chromatography using Source Q anion resin was applied, and high-purity rMeV N protein was harvested. To characterize the similarity with the native viral protein to purified N protein, structural mimicry of purified recombinant proteins with intact rMeV N was shown through transmission electron microscopy, and the truncation and the phosphorylation status of the expressed protein were analyzed. These results suggest that the rMeV N purified by ion-exchange chromatography has features similar to those of naïve N including a self-assembled structure, phosphorylation and antigenic function. Thus, these expression and purification methods can be applied to the large-scale production of the rMeV N, which is essential for the development of new diagnostic tools and vaccines for acute and chronic MeV infections.

An alternative easy method for antibody purification and analysis of protein–protein interaction using GST fusion proteins immobilized onto glutathione–agarose

Immobilization of small proteins designed to perform protein-protein assays can be a difficult task. Often, the modification of reactive residues necessary for the interaction between the immobilized protein and the matrix compromises the interaction between the protein and its target. In these cases, glutathione-S-transferase (GST) is a valuable tag providing a long arm that makes the bait protein accessible to the mobile flow phase of the chromatography. In the present report, we used a GST fusion version of the 8-kDa protein serine protease inhibitor Kazal-type 3 (SPINK3) as the bait to purify anti-SPINK3 antibodies from a rabbit crude serum. The protocol for immobilization of GST-SPINK3 to glutathione-agarose beads was modified from previously reported protocols by using an alternative bifunctional cross-linker (dithiobis(succinimidyl propionate)) in a very simple procedure and by using simple buffers under physiological conditions. We concluded that the immobilized protein remained bound to the column after elution with low pH, allowing the reuse of the column for alternative uses, such as screening for other protein-protein interactions using SPINK3 as the bait.

High yield purification of nanobodies from the periplasm of E. coli as fusions with the maltose binding protein

Nanobodies (Nbs) are single domain antibodies based on the variable domains of heavy chain only antibodies (HCAbs) found in camelids, also referred to as VHHs. Their small size (ca. 12–15kDa), superior biophysical and antigen binding properties have made Nbs very attractive molecules for multiple biotechnological applications, including human therapy. The most widely used system for the purification of Nbs is their expression in the periplasm of Escherichia coli with a C-terminal hexa-histidine (His6) tag followed by immobilized metal affinity chromatography (IMAC). However, significant variability in the expression levels of different Nbs are routinely observed and a single affinity chromatography step is often not sufficient to obtain Nbs of high purity. Here, we report an alternative method for expression and purification of Nbs from the periplasm of E. coli based on their fusion to maltose binding protein (MBP) in the N-terminus and His6 tag in the C-terminus (MBP-NbHis6). Soluble MBP-NbHis6 fusions were consistently expressed at high levels (⩾12mg/L of induced culture in shake flasks) in the periplasm of E. coli HM140, a strain deficient in several periplasmic proteases. Highly pure MBP-NbHis6 fusions and free NbHis6 (after site specific proteolysis of the fusions), were recovered by amylose and metal affinity chromatography steps. The monomeric nature of the purified NbHis6 was determined by gel filtration chromatography. Lastly, we demonstrated by ELISA that both monomeric NbHis6 and MBP-NbHis6 fusions retained antigen binding activity and specificity, thus facilitating their direct use in antigen recognition assays.

An affinity chromatography method used to purify His-tag-displaying bio-nanocapsules

A bio-nanocapsule derived from the hepatitis B virus (HBV) is expected to be useful as a drug delivery system carrier. Because various types of bio-nanocapsules have been developed, a simple and versatile purification method for bio-nanocapsules would be useful. Therefore, this study was focused on establishing a simple purification method using affinity chromatography by inserting a histidine tag (His-tag) into a bio-nanocapsule. The method achieved a simple, one-step purification with a yield that was 2.5-fold higher than conventional ultracentrifugation, and thus would be a desirable alternative method for recombinant virus-like particle purification.

Economical method for midiprep plasmid DNA purification using diatomaceous earth

Diatomaceous earth has been used for DNA purification as it can produce high quality plasmid DNA. We describe an alternative and economical method for plasmid DNA purification from a 50-ml bacterial culture using diatomaceous earth, plastic pipette tips, conical tubes, centrifuges, and without requiring a vacuum system. Depending on the plasmid size and the origin of replication, this method yielded 200-800 µg plasmids with an A260/A280 of 1.8-2.0. These purified plasmids are suitable for many applications such as DNA sequencing and transfection experiments.

Adsorption performance of titanium dioxide (TiO2) coated air filters for volatile organic compounds

The photocatalytic oxidation (PCO) technology as an alternative method for air purification has been studied for decades and a variety of PCO models indicate that the adsorption of reactants on the catalyst surface is one of the major physical and chemical processes occurring at a heterogeneous photocatalytic reaction. However, limited study explored the adsorption effect of a photocatalyst. This study carried out a systematic evaluation of adsorption performance of titanium dioxide (TiO2) coated fiberglass fibers (FGFs), TiO2 coated carbon cloth fibers (CCFs), and original CCFs air filters at various relative humidity conditions for nine volatile organic compounds. TiO2/FGFs, TiO2/CCFs, and CCFs were characterized by SEM for morphology and N2 adsorption isotherm for BET surface area and pore structure. A bench-scale adsorption test setup was constructed and adsorption tests were performed at various relative humidity conditions and four different injected concentrations for each compound. The isothermal adsorption curves at low concentration levels were obtained and they were well described by Langmuir isotherm model. It was noticed that there were significant differences between the adsorption behaviors and photocatalytic activities of TiO2/FGFs and TiO2/CCFs. It was concluded that adsorption performance is closely related to the characteristics of substrates and therefore, the development of a substrate with high adsorption ability is a promising trend for improving the performance of the UV-PCO technology.

Different Purification Approaches for the Integral Membrane Protein EmrE Leads to Biochemical and Biophysical Differences in the Protein

EmrE is an integral membrane protein in Escherichia coli belonging to a class of multidrug resistance transporters: the Small Multidrug Resistance transporter family (SMR). EmrE effluxes quaternary cationic compounds (QCC) using a proton motive force. Many groups studying this protein utilize a C-terminal myc-eptitope- 6 histidine tag engineered into EmrE for the purpose of protein purification in a detergent-containing aqueous environment. An alternative method for EmrE purification involves using a solvent mixture of chloroform:methanol:water to extract the protein from bacterial membranes. The EmrE isolated using the latter method does not have any tag. As there is different data appearing from different labs, the goal of this research is to compare the protein purified in with the different approaches analyzed under identical conditions. Untagged and tagged EmrE was analyzed and compared by fluorescence spectroscopy. Fluorescence emission spectra of untagged EmrE revealed a lower combined fluorescence intensity of the tyrosine and tryptophan fluorescence compared to tagged EmrE. A red edge excitation shift (REES) effect was also observed for tagged EmrE indicating that there is a population of fluorophores that have a slower rate of solvent relaxation, unlike the fluorophores in untagged EmrE. Functional analysis of the EmrE by QCC ligand-binding (Ethidium, methyl viologen, cetylpyridinium) was investigated using fluorescence quenching binding curves. Untagged EmrE and tagged EmrE binding curves with ethidium were similar, whereas binding of the methyl viologen and cetylpyridinium were different between untagged and tagged EmrE. These results show that the two purification approaches lead to differences in the structural and function states of this integral membrane protein.

Development of a novel rDNA based plasmid for enhanced cell surface display on Yarrowia lipolytica.

In this study, a novel rDNA based plasmid was developed for display of heterologous proteins on the cell surface of Yarrowia lipolytica using the C-terminal end of the glycosylphosphatidylinositol (GPI) anchored Y. lipolytica cell wall protein 1 (YlCWP1). mCherry was used as a model protein to assess the efficiency of the constructed plasmid. Y. lipolytica transformants harbouring the expression cassettes showed a purple colour phenotype on selective YNB-casamino plates as compared to control cells indicating that mCherry was displayed on the cells. Expression of mCherry on cells of Y. lipolytica was confirmed by both fluorescent microscopy and flow cytometry. Furthermore, SDS-PAGE analysis and matrix-assisted laser desorption/ionization (MALDI)-time-of (TOF)-mass spectrometry (MS) peptide mass fingerprinting (PMF) confirmed that the protein cleaved from the yeast cells using enterokinase was mCherry. Efficient cleavage of mCherry reported in this work offers an alternative purification method for displayed heterologous proteins on Y. lipolytica cells using the plasmid constructed in this study. The developed displaying system offers great potential for industrial production and purification of heterologous proteins at low cost.

An Alternative Purification Method for Human Serum Paraoxonase 1 and its Interactions with Sulfonamides

Paraoxonase 1 (PON1), a high-density lipoprotein (HDL)-associated esterase, is known to mediate antioxidant and antiatherogenic properties. Purification of PON1 has been challenging for a long time. Here, we report a novel purification technique for this enzyme, which allowed us to obtain human serum paraoxonase 1 (hPON1) using straightforward chromatographic methods, such as Diethylaminoethyl-Sephadex anion exchange chromatography and Sepharose 4B-4-phenylazo-2-naphthaleneamine hydrophobic interaction chromatography. We purified the enzyme 302-fold with a final specific activity of 4775 U/mg and a yield of 32%. Furthermore, we examined the in vitro effects of some sulfonamide derivatives, such as sulfacetamide, homosulfanilamide (mafenide), sulfosalazine, furosemide, acetazolamide, and 1,3,4-thiadiazole-2-sulfonamide on the enzyme activity to better understand the inhibitory properties of the molecules. The six sulfonamides dose-dependently decreased the activity of hPON1 with inhibition constants in the millimolar-micromolar range. This study provides an efficient method, which may be useful for other enzymes such as those related to acetylcholinesterase. It also demonstrates the off-target activity of sulfonamides.

Isolation of Fungal Homokaryotic Lines from Heterokaryotic Transformants by Sonic Disruption of Mycelia

Fungal hyphae--and in some cases, spores--are multi-nucleate. During genetic transformation of these spores or mycelia, only one nucleus generally receives the transferred T-DNA generating heterokaryotic colonies. Characterization of genetic changes, such as the effects of gene disruption in the transformants, requires purified homokaryotic lines. Hyphal tip transfer has conventionally been used to isolate homokaryons. We developed an alternative method for purification of fungal homokaryons from transformed heterokaryotic lines of Sclerotinia sclerotiorum. Ultrasound pulses were used to generate bi-septate, unicellular hyphal fragments that regenerate under selection to produce homokaryotic lines that can be easily identified using colony PCR. This technique facilitates the purification of transformed lines, which allows for routine genome manipulation, and should be adaptable for other filamentous fungi.

Formation of PCDD and PCDF in the thermal treatment of footwear leather wastes

The leather waste generated by the footwear industry is considered dangerous due to the presence of trivalent chromium, derived from the salt utilized to tan hides. In Brazil, the majority of this waste is disposed on landfills and only about 3% are recycled. The thermal treatment is an alternative method for purification of such residues. By using this technique it is possible to generate energy and recover the chromium present in the ash for the production of basic chromium sulfate (tanning industry), high carbon ferrochromium or carbon-free ferrochromium (steel industry). In the last 10 years, the gasification and combustion of footwear leather waste have been intensively studied at the Federal University of Rio Grande do Sul. The research experiment for characterization of the emissions of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F) were carried out in a semi-pilot unit (350 kW th). From new investments the thermal capacity of the unit will increase to 600 kW th. The unit will produce power from the heat generated in the combustion. The experimental results indicated that during the thermal treatment of footwear leather wastes, the formation mechanism of PCDD/F is the de novo synthesis. Most of PCDD/F were found in the particulate phase (>95%). A kinetic model was used for discussion of the achieved experimental results. The model is based in the carbon gasification, PCDD/F formation, desorption and degradation. From the conclusions obtained in this work will be possible minimize the PCDD/F formation in process of combustion of footwear leather wastes.

Purification of high-throughput organic synthesis libraries by counter-current chromatography

Experiments were performed to evaluate whether counter-current chromatography (CCC) could function as an alternative purification method to reversed-phase high-performance liquid chromatography (RP-HPLC) and normal-phase supercritical fluid chromatography (SFC). RP-HPLC and SFC are the routine methods currently used in our high-throughput purification (HTP) facility for the purification of high-throughput organic synthesis (HTOS) libraries and medicinal chemistry reaction mixtures. Pre-equilibration of the solvent mixture layers was not mandatory for effective chromatography when hexanes–ethyl acetate–methanol–water (HEMW) solvent mixtures were used. Key to the use of CCC for high-throughput applications is the ability to effectively select a solvent system appropriate to each library member. Pilot-scale CCC elution time was used to estimate a starting solvent ratio and RP-HPLC retention time was then used to adjust solvent ratios within a particular library. It was also found that dimethyl sulfoxide (DMSO) and DMSO–methanol were suitable as sample injection solvents when using the HEMW solvent systems.

Purification and analysis of polyhistidine-tagged human parvovirus B19 VP1 and VP2 expressed in insect cells

Human parvovirus B19 is an autonomously replicating human pathogen with a specific tropism for human erythroid progenitor cells. There is an interest in producing empty nucleocapsids of B19 as they can be used as tools in molecular biology and diagnostics. Native B19 virus particles are formed from two structural viral proteins, VP1 and VP2. The VP2 protein alone is able to self assemble and consequently form virus-like particles (VLPs) in heterologous expression systems. Purification of recombinant VLPs has been conducted using various traditional methods. These include laborious and time-consuming, e.g. cesium chloride or sucrose gradient ultracentrifugation steps, allowing limited working volumes to be processed. Therefore, an alternative purification method enabling process scale-up was developed and evaluated. Polyhistidine-tagged versions of B19 VP1 and VP2 capsid proteins were engineered and produced using the baculovirus expression system. The recombinant protein products were purified by immobilized metal-ion affinity chromatography (IMAC) and analyzed by SDS-PAGE, immunoblotting, electron microscopy, and enzyme-linked immunosorbent assays. Further, the immunological properties of the recombinant proteins were evaluated. The results showed that the VP2 fusion protein assembled into capsid-like structures and that both VP1 and VP2 following purification by IMAC have potential as antigens for diagnosis of a B19 infection.

Cell surface display of functionally active lipases from Yarrowia lipolytica in Pichia pastoris

The lipase genes of Yarrowia lipolytica, LIPY7 and LIPY8, fused with FLO—flocculation domain sequence from Saccharomyces cerevisiae at their N-termini, were expressed in Pichia pastoris KM71. Following the induction with methanol, the recombinant proteins were displayed on the cell surface of P. pastoris, as confirmed by the confocal laser scanning microscopy. The LipY7p and LipY8p were anchored on P. pastoris via the flocculation functional domain of Flo1p. The surface-displayed lipases were characterized for their application as the whole-cell biocatalyst. These lipases can also be cleaved off from their anchor by enterokinase treatment to yield functionally active proteins in the supernatant offering an alternative purification method for LipY7p and LipY8p.

An alternate high yielding purification method for Clitoria ternatea lectin

In our previous publication we had reported the purification and characterization of Clitoria ternatea agglutinin from its seeds on fetuin CL agarose affinity column, designated CTA [A. Naeem, S. Haque, R.H. Khan. Protein J., 2007]. Since CTA binds β- d-galactosides, this lectin can be used as valuable tool for glycobiology studies in biomedical and cancer research. So an attempt was made for a high yielding alternative purification method employing the use of asialofetuin CL agarose column for the above-mentioned lectin, designated CTL. The fetuin affinity purified agglutinin was found similar to asialofetuin affinity purified lectin in SDS pattern, HPLC and N-terminal sequence. The content of lectin was found to be 30 mg/30 g dry weight of pulse. The yield was 2.8% as compared to 0.3% obtained on fetuin column. The number of tryptophan and tyrosine estimated was four and six per subunit.