Simple Purification Procedure Research Articles

Polyethyleneimine-Supported Triphenylphosphine and Its Use as a Highly Loaded Bifunctional Polymeric Reagent in Chromatography-Free One-Pot Wittig Reactions

A polyethyleneimine-supported triphenylphosphine reagent has been synthesized and used as a highly loaded bifunctional homogeneous reagent in a range of one-pot Wittig reactions that afforded high yields of the desired products after simple purification procedures. The approach also served efficiently in tandem reaction sequences involving a one-pot Wittig reaction followed by conjugate reduction of the newly formed alkene product in situ. In these transformations, the phosphine oxide groups generated in the Wittig reaction served as the catalyst for activating trichlorosilane in the subsequent reduction reaction.

Discovery and Biochemical Characterization of UDP-Glucose Dehydrogenase from Granulibacter bethesdensis.

UDP-glucose dehydrogenases (EC 1.1.1.22) are responsible for the conversion of UDP-glucose to UDP-glucuronic acid, a key precursor in the biosynthesis of glycoconjugates. Herein we report the discovery and characterization of a UDPglucose dehydrogenase (GbUGD) from Granulibacter bethesdensis, a bacterium originally isolated from the lymph nodes of patients with chronic granulomatous disease (CGD). The recombinant form of the protein was expressed in high yield and the purified enzyme showed highest activity at 37°C/pH 9.0 and was strongly inhibited by Zn(2+) ions, sodium dodecyl sulfate (SDS) and urea. UDP-xylose, an allosteric feedback inhibitor, reduced significantly the activity of the enzyme. High activities were observed using the co-substrates UDP-glucose and NAD+, whereas no activity could be detected using other nucleotide sugars or NADP(+) as potential alternative substrates. The high activity combined with the simple purification procedure used make GbUGD a valuable new alternative biocatalyst for the synthesis of UDP-glucuronic acid or the development of NAD+ regeneration systems.

New extractant N,N′-dimethyl-N,N′-dicyclohexyl-2,(2′-dodecyloxyethyl)-malonamide (DMDCDDEMA) for radiotoxic acidic waste remediation: Synthesis, extraction and supported liquid membrane transport studies

N,N′-dimethyl-N,N′-dicyclohexyl-2,(2′-dodecyloxyethyl)-malonamide (DMDCDDEMA) has been synthesized by a simpler route; with a better yield, than conventional pentaalkyl substituted diamide synthesis procedures. This route involves simple purification procedures like crystallization leading to desired molecule with high purity. The key step of 1-tosyloxy-3-oxa-pentadecane synthesis has been achieved by selective monoprotection of glycol with simpler chemical transformations. The synthesized product has been tested for radiotoxic metal ions (such as 241Am, 237Np, 239Pu, 233U) extraction and their transport behavior across a supported liquid membrane from nitric acid solutions. The effects of various experimental parameters on the possible radiotoxic waste remediation by this diamide have been evaluated quantitatively. The performance of DMDCDDEMA has been evaluated vis-à-vis other proposed carrier extractants for actinide partitioning under conditions of study. The study suggests that the relatively easier synthesis and its application in membrane based separations may help in scale up operations in radiotoxic waste remediation.

Cloning, expression, and purification of insect (Sitophilus oryzae) alpha-amylase, able to digest granular starch, in Yarrowia lipolytica host.

Raw-starch-digesting enzymes (RSDE) are of major importance for industrial applications, as their usage greatly simplifies the starch processing pipeline. To date, only microbial RSDE have gained considerable attention, since only microbial production of enzymes meets industrial demands. In this study, α-amylase from rice weevil (Sitophilus oryzae), the major rice pest, was cloned and expressed in Yarrowia lipolytica Po1g strain. The enzyme was secreted into the culture medium, and the peak activity (81 AU/L) was reached after only 29 h of culturing in 5-L bioreactors. Through simple purification procedure of ammonium sulfate precipitation and affinity chromatography, it was possible to purify the enzyme to apparent homogeneity (25-fold purification factor, at 5 % yield). The optimal conditions for the α-amylase activity were pH 5.0 and a temperature of 40 °C. The α-amylase studied here did not show any obligate requirement for Ca2+ ions. The recombinant α-amylase appeared to efficiently digest granular starch from pea, amaranth, waxy corn, and waxy rice.

Purification and characterization of glucose-6-phosphate dehydrogenase from camel liver.

Glucose-6-phosphate dehydrogenase from camel liver was purified to homogeneity by ammonium sulfate precipitation and a combination of DEAE-cellulose, Sephacryl S-300 gel filtration, and 2′, 5′ ADP Sepharose 4B affinity chromatography columns. The specific activity of camel liver G6PD is increased to 1.80438 units/mg proteins with 63-fold purification. It turned out to be homogenous on both native PAGE and 12% SDS PAGE, with a molecular weight of 64 kDa. The molecular weight of the native form of camel liver G6PD was determined to be 194 kDa by gel filtration indicating a trimeric protein. The K m value was found to be 0.081 mM of NADP+. Camel liver G6PD displayed its optimum activity at pH 7.8 with an isoelectric point (pI) of pH 6.6–6.8. The divalent cations MgCl2, MnCl2, and CoCl2 act as activators; on the other hand, CaCl2 and NiCl2 act as moderate inhibitors, while FeCl2, CuCl2, and ZnCl2 are potent inhibitors of camel liver G6PD activity. NADPH inhibited camel liver G6PD competitively with K i value of 0.035 mM. One binding site was deduced for NADPH on the enzyme molecule. This study presents a simple and reproducible purification procedure of G6PD from the camel liver.

Synthesis of S-adenosyl-L-methionine in Escherichia coli

Abstract S-adenosyl-L-methionine (SAM) is an important physiological metabolite in vivo and may be useful in medicines. SAM is produced from L-methionine and ATP catalyzed by S-adenosyl-L-methionine synthetase (SAMS) in vivo. In this study, the gene encoding SAMS was cloned and a genetically engineered Escherichia coli (E. coli) BL21(pET-28a-SAMS) was constructed. The recombinant SAMS with a molecular mass of approximately 46 kDa was expressed by inducing the engineered E. coli using isopropyl-s-D-1-thiogalactopyranoside (IPTG) as an inducer. To produce SAM using a low-cost, nontoxic and highperformance expression system, lactose was used as a substitute for IPTG to induce BL21(pET-28a-SAMS). By optimizing the expression conditions, the concentration of SAM produced by the engineered E. coli was 48 mg/L in the culture medium supernatant. To increase the concentration of SAM produced, a coupled system was constructed consisting of E. coli BL21(pET-28a-SAMS) and Saccharomyces cerevisiae (S. cerevisiae) JM-310. In this coupled system, ATP generated from S. cerevisiae was provided to E. coli for producing a higher concentration of SAM. The SAM concentration in the coupled system reached 1.7 g/L. SAM was purified by a weak acid cationic exchange resin D113, and a simple and economical purification procedure for SAM isolation was achieved. SAM was confirmed by High Performance Liquid Chromatography-tandem Mass Spectrometry analysis. Our study provides a feasible and convenient approach to produce SAM.

High-yield preparation of graphene oxide from small graphite flakes via an improved Hummers method with a simple purification process

Graphite flakes with sizes in the range of 3–20μm can be completely converted into single-layer graphene oxide (GO) with a high yield of 171±4% via an improved Hummers method. This yield is much higher than that (110±3%) from larger graphite flakes with sizes of 10–100μm. The structures, qualities and size distributions of the GO sheets prepared from both graphite precursors are nearly identical. More importantly, in the case of using small graphite flakes, the GO dispersion can be directly purified without the requirement of centrifugation to remove unoxidized residuals. Considering the high yield, simplified purification procedure, and high-quality of GO, the modification of using graphite flakes with small sizes is an important step towards the mass-production of GO at industrial scale.

Isolation of plasmodesmata from Arabidopsis suspension culture cells.

Due to their position firmly anchored within the plant cell wall, plasmodesmata (PD) are notoriously difficult to isolate from plant tissue. Yet, getting access to isolated PD represents the most straightforward strategy for the identification of their molecular components. Proteomic and lipidomic analyses of such PD fractions have provided and will continue to provide critical information on the functional and structural elements that define these membranous nano-pores. Here, we describe a two-step simple purification procedure that allows isolation of pure PD-derived membranes from Arabidopsis suspension cells. The first step of this procedure consists in isolating cell wall fragments containing intact PD while free of contamination from other cellular compartments. The second step relies on an enzymatic degradation of the wall matrix and the subsequent release of "free" PD. Isolated PD membranes provide a suitable starting material for the analysis of PD-associated proteins and lipids.

Functionalized spider silk spheres as drug carriers for targeted cancer therapy.

Bioengineered spider silk is a biomaterial that combines the properties of self-assembly, biocompatibility and biodegradability with reasonable accessibility and a simple purification procedure. Moreover, genetic engineering enables the functionalization of silk by adding the peptide coding sequences of the desired attribute. Hybrids composed of Her2 binding peptides (H2.1 or H2.2) and bioengineered silk MS1 (based on the MaSp1 sequence from N. clavipes) were designed. Bioengineered silks were expressed in a bacterial system and purified using a tag-free thermal method. The hybrid silks with N-terminal functionalization were bound more efficiently to cells that were overexpressing Her2 than those with the C-terminal fusion. Moreover, the functionalization did not impede the self-assembly property of bioengineered silk, enabling the processing of silk proteins into spheres. The binding domains were exposed on the surface of the spheres, because the functionalized particles specifically bound and internalized into Her2-overexpressing cells. The binding of the functionalized spheres to Her2-positive cells was significantly higher compared with the control sphere and Her2-negative cell binding. Silk spheres were loaded with doxorubicin and showed pH-dependent drug release. The silk spheres were not cytotoxic, unless they were loaded with the drug doxorubicin. This study indicates the ability of drug-loaded functionalized spider silk spheres to serve as a carrier for targeted cancer therapy.

A novel fed-batch based strategy for enhancing cell-density and recombinant cyprosin B production in bioreactors.

Nowadays, the dairy industry is continuously looking for new and more efficient clotting enzymes to create innovative products. Cyprosin B is a plant aspartic protease characterized by clotting activity that was previously cloned in Saccharomyces cerevisiae BJ1991 strain. The production of recombinant cyprosin B by a batch and fed-batch culture was compared using glucose and galactose as carbon sources. The strategy for fed-batch cultivation involved two steps: in the first batch phase, the culture medium presented glucose 1% (w/v) and galactose 0.5% (w/v), while in the feed step the culture medium was constituted by 5% (w/v) galactose with the aim to minimize the GAL7 promoter repression. Based on fed-batch, in comparison to batch growth, an increase in biomass (6.6-fold), protein concentration (59%) and cyprosin B activity (91%) was achieved. The recombinant cyprosin B was purified by a single hydrophobic chromatography, presenting a specific activity of 6×10(4) U·mg(-1), corresponding to a purification degree of 12.5-fold and a recovery yield of 16.4%. The SDS-PAGE analysis showed that recovery procedure is suitable for achieving the purified recombinant cyprosin B. The results show that the recombinant cyprosin B production can be improved based on two distinct steps during the fed-batch, presenting that this strategy, associated with a simplified purification procedure, could be applied to large-scale production, constituting a new and efficient alternative for animal and fungal enzymes widely used in cheese making.

High-level production of Fc-fused kringle domain in Pichia pastoris

Recently, as a new non-immunoglobulin-based protein scaffold, a human kringle domain was successfully engineered toward biologically functional agonists and antagonists. In this study, the fed-batch cultivation conditions were optimized for enhanced production of an Fc-fused kringle domain (KD548-Fc) in Pichia pastoris. Fed-batch cultivations were performed in 5-l laboratory-scale bioreactors, and in order to find the optimal conditions for high-level production of KD548-Fc, several parameters including the initial carbon source (glycerol) concentration, temperature, and pH were investigated. When cells were cultivated at pH 4.0 and 25 °C with 9.5 % glycerol in the initial medium, the highest production yield (635 mg/l) was achieved with high productivity (7.2 mg/l/h). Furthermore, functional KD548-Fc was successfully purified from the culture broth using a simple purification procedure with high purity and recovery yield.

Exploitation of FTA Cartridges for the Sampling, Long-Term Storage, and DNA-Based Analyses of Plant-Parasitic Nematodes

The use of DNA-based analyses in molecular plant nematology research has dramatically increased over recent decades. Therefore, the development and adaptation of simple, robust, and cost-effective DNA purification procedures are required to address these contemporary challenges. The solid-phase-based approach developed by Flinders Technology Associates (FTA) has been shown to be a powerful technology for the preparation of DNA from different biological materials, including blood, saliva, plant tissues, and various human and plant microbial pathogens. In this work, we demonstrate, for the first time, that this FTA-based technology is a valuable, low-cost, and time-saving approach for the sampling, long-term archiving, and molecular analysis of plant-parasitic nematodes. Despite the complex structure and anatomical organization of the multicellular bodies of nematodes, we report the successful and reliable DNA-based analysis of nematode high-copy and low-copy genes using the FTA technology. This was achieved by applying nematodes to the FTA cards either in the form of a suspension of individuals, as intact or pestle-crushed nematodes, or by the direct mechanical printing of nematode-infested plant tissues. We further demonstrate that the FTA method is also suitable for the so-called "one-nematode-assay", in which the target DNA is typically analyzed from a single individual nematode. More surprisingly, a time-course experiment showed that nematode DNA can be detected specifically in the FTA-captured samples many years after initial sampling occurs. Collectively, our data clearly demonstrate the applicability and the robustness of this FTA-based approach for molecular research and diagnostics concerning phytonematodes; this research includes economically important species such as the stem nematode (Ditylenchus dipsaci), the sugar beet nematode (Heterodera schachtii), and the Northern root-knot nematode (Meloidogyne hapla).

Thin film microfluidic synthesis of fluorescent highly substituted pyridines

Fluorescent polysubstituted pyridines are readily accessible as a single process using a thin film vortex fluidic device, with the compounds obtained in good yield following simple purification procedures.

Characterization of UO22+binding to osteopontin, a highly phosphorylated protein: insights into potential mechanisms of uranyl accumulation in bones

Bones are one of the few organs in which uranyl (UO2(2+)) accumulates. This large dioxo-cation displays affinity for carboxylates, phenolates and phosphorylated functional groups in proteins. The noncollagenous protein osteopontin (OPN) plays an important role in bone homeostasis. It is mainly found in the extracellular matrix of mineralized tissues but also in body fluids such as milk, blood and urine. Furthermore, OPN is an intrinsically disordered protein, which, like other proteins of the SIBLING family, contains a polyaspartic acid sequence and numerous patterns of alternating acidic and phosphorylated residues. All these properties led to the hypothesis that this protein could be prone to UO2(2+) binding. In this work, a simple purification procedure enabling highly purified bovine (bOPN) and human OPN (hOPN) to be obtained was developed. Various biophysical approaches were set up to study the impact of phosphorylations on the affinity of OPN for UO2(2+) as well as the formation of stable complexes originating from structural changes induced by the binding of this metal cation. The results obtained suggest a new mechanism of the interaction of UO2(2+) with bone metabolism and a new role for OPN as a metal transporter.

A His6-SUMO-eXact tag for producing human prepro-Urocortin 2 in Escherichia coli for raising monoclonal antibodies

This is a first report of recombinant production of human prepro-Urocortin 2 in Escherichia coli by N-terminal fusion with a triple His6-SUMO-eXact tag and its subsequent use as an antigen for the production and screening of very high affinity monoclonal antibodies. The rationale for this combinatorial construct is that the His tag allows first step protein purification of insoluble and soluble proteins, the SUMO tag enhances protein expression level and solubility, while the eXact tag facilitates anion-triggered on-column cleavage of the triple tag to recover pure native proteins in a simple two-step protein purification procedure. Compared with an eXact fusion alone, the presence of the SUMO moiety enhanced overall expression levels by 4 to 10 fold but not the solubility of the highly basic prepro-Urocortin 2. Insoluble SUMO-eXact-preproUCN2 was purified in milligram quantities by denaturing IMAC and solubilized in native phosphate buffer by on-column refolding or step-wise dialysis. Only a small fraction of this solubilized protein was able to bind onto the eXact™ affinity column and cleaved by NaF treatment. To test whether binding and cleavage failure was due to improperly refolded SUMO-eXact-preproUCN2 or to the presence of N- and C-terminal sequences flanking the eXact moiety, we created a SUMO-eXact-thioredoxin construct which was overexpressed mainly in the soluble form. This protein bound to and was cleaved efficiently on the eXact™ column to yield native thioredoxin. Solubilized SUMO-eXact-preproUCN2 was used successfully to generate two high affinity mouse monoclonal antibodies (KD~10−10 and 10−11M) specific to the pro-region of Urocortin 2.

An Efficient Solvent-Free Synthesis of Naphthopyranopyrimidines Using Heteropolyacid as an Ecofriendly Catalyst

An efficient, one-pot, three-component synthesis of naphthopyranopyrimidines using β-naphthol, aldehydes, and 6-amino-1,3-dimethyl uracil catalyzed by heteropolyacid has been achieved within a short period of time. The present methodology offers several advantages such as ecofriendly catalyst, low catalyst loading, short reaction time, simple purification procedure, and excellent yields.

High-level secretory production of recombinant single-chain variable fragment (scFv) in Corynebacterium glutamicum

We describe the development of a new secretory production system for the enhanced production of a single-chain variable fragment (scFv) against the anthrax toxin in Corynebacterium glutamicum. For efficient secretory production of the antibody fragment, the following components were examined: (1) signal peptides, (2) codon usage of antibody fragment, (3) promoters, (4) 5' untranslated region (5' UTR) sequence, and (5) transcriptional terminator. Among all the systems examined, the use of a codon-optimized gene sequence, a Sec-dependent PorB signal peptide, and a fully synthetic H36 promoter, allowed the highest production of antibody fragments in a culture medium. For large-scale production, fed-batch cultivations were also conducted in a 5-L lab-scale bioreactor. When cells were cultivated in semi-defined media, cells could grow up to an OD600 of 179 for 32 h and an antibody fragment concentration as high as 68 mg/L could be obtained in a culture medium with high purity. From the culture medium, the secreted antibody was successfully purified using a simple purification procedure, with correct binding activity confirmed by enzyme-linked immunosorbent assay. To the best of our knowledge, this is the first report of a fed-batch cultivation for antibody fragment production in C. glutamicum.

Fermentation in vitro of EGCG, GCG and EGCG3"Me isolated from Oolong tea by human intestinal microbiota

In the present study, EGCG, GCG and EGCG3"Me were prepared from Chinese Oolong tea by extraction with hot water and simple purification procedures of polyamide column chromatography and Toyopearl HW-40S column chromatography. Then, the effects of EGCG, GCG and EGCG3"Me on human intestinal microbiota in vitro were evaluated by monitoring the bacterial populations and analyzing the concentrations of short-chain fatty acids (SCFA) at different fermentation times. As a result, EGCG, GCG and EGCG3"Me promoted the growth of Bifidobacterium spp. and Lactobacillus/Enterococcus groups and exhibited inhibitory effects on the growth of Bacteroides–Prevotella, Clostridium histolyticum and Eubacterium–Clostridium groups, while they did not affect the population of total bacteria. Moreover, the total SCFA concentrations in cultures with EGCG, GCG and EGCG3"Me were relatively higher than that of the control. Together, these results suggest that EGCG, GCG and EGCG3"Me have potential prebiotic-like activity by modulating intestinal microbiota and generating SCFA, contributing to the improvements of host health.

Hyper secretion of Thermobifida fusca β-glucosidase via a Tat-dependent signal peptide using Streptomyces lividans

BackgroundProtein production as secretory-form is a powerful tool in industrial enzyme production due to the simple purification procedure. Streptomyces lividans is a versatile host for secretory production of useful proteins. In order to expand the amount of secreted protein, signal peptide sequences, which encourage protein secretion from inside cell to extracellular environment, are one of the most significant factors. In this study, we focused on Streptomyces lividans as a host strain to secrete useful proteins, and screened for signal peptides from the biomass-degradation enzymes derived from Thermobifida fusca YX and S. lividans.ResultsThree candidate signal peptides were isolated and evaluated for their protein secretion ability using β-glucosidase derived from T. fusca YX, which is a non-secreted protein, as a model protein. Using S. lividans xylanase C signal peptide, the amount of produced the β-glucosidase reached 10 times as much as that when using Streptomyces cinnamoneus phospholipase D signal peptide, which was identified as a versatile signal peptide in our previous report. In addition, the introduction of the β-glucosidase fused to xylanase C signal peptide using two kinds of plasmid, pUC702 and pTYM18, led to further protein secretion, and the maximal level of produced the β-glucosidase increased up to 17 times (1.1 g/l) compared to using only pUC702 carrying the β-glucosidase fused to S. cinnamoneus phospholipase D signal peptide.ConclusionIn the present study, we focused on signal peptide sequences derived from biomass degradation enzymes, which are usually secreted into the culture supernatant, and screened for signal peptides leading to effective protein secretion. Using the signal peptides, the hyper-protein secretion system was successfully demonstrated for the cytoplasmic β-glucosidase.

Direct (Hetero)arylation Polymerization: An Effective Route to 3,4-Propylenedioxythiophene-Based Polymers with Low Residual Metal Content.

We report the use of direct (hetero)arylation polymerizations (DHAP) as a means of obtaining 3,4-propylenedioxythiophene-based conjugated polymers for use in electrochromics. This method offers a rapid route to achieving polymers in high yields with simplified purification procedures and low residual metal content, as determined by inductive coupled plasma-mass spectrometry (ICP-MS). The studied polymers possess comparable electrochromic properties to those previously reported by our group, implying that their switching ability from a colored to a transmissive state is independent of the residual metallic impurities.