Large-scale Purification Procedure Research Articles

Purification of polysaccharide from Lentinus edodes water extract by membrane separation and its chemical composition and structure characterization

Currently, most of the polysaccharide fractions are purified using the methods usually limited to the laboratory scale or easily raising question of residual reagent, which may obstruct the future application of polysaccharide. In our study, we employed sequential ultrafiltration with different membranes combined with HPGPC to purify polysaccharide fractions from the water extract of Lentinus edodes directly. The chemical composition, morphology and structure characteristic were analyzed by chemical methods and instrumental analysis including automatic amino acid analyzer, Elemental Analyzer, SEM, HPGPC, HPAEC-PAD, FT-IR and NMR. Results indicated that three polysaccharide fractions named as LE-UF-1, LE-UF-2 and LE-UF-3 were successfully purified using ultrafiltration membranes with MWCO of 2.5, 5 and 10 kDa in sequence. Mw and polysaccharide content of LE-UF-1 (~136 kDa, 86.11%) were the highest, followed by LE-UF-2 (14–61 kDa, 49.82%) and LE-UF-3 (14–35 kDa, 22.44%). Surprisingly, LE-UF-2 had 11.73% amino acid, and LE-UF-3 had 19.64% amino acid. The polysaccharide in all the three fractions was heteropolysaccharide that mainly consisted of Glc, Gal and Man. There might be more β-sugar residues in LE-UF-1 and LE-UF-2, while more α-sugar residues in LE-UF-3. Our study provides a greener and more efficient potential procedure for large-scale polysaccharide purification, and is able to prepare the polysaccharide fractions from Lentinus edodes that may be used for food or drug development.

Water Extraction of Anthocyanins from Black Rice and Purification Using Membrane Separation and Resin Adsorption

A simple and efficient method to extract and purify anthocyanins from black rice was developed in this work. After pretreatment of black rice, anthocyanins were extracted by buffer solutions, and were purified using membrane separation and resin adsorption. The single-factor experiments and orthogonal design were applied. The results showed that extraction ratio could be 0.99 ± 0.01% under the optimal condition of liquid–solid ratio 12:1, temperature 50C, time 80 min and pH 3.2. The 3 kDa molecular weight cut-off (MWCO) was used to remove proteins and polysaccharides, and the DM301 resin was found to have both good adsorptivity and desorptivity for anthocyanins. After the elution using 85% (vol/vol) ethanol solution, the purified anthocyanins were obtained by retention with the membrane of 200 Da MWCO. The recovery and purity of the anthocyanins finally obtained was 0.86% and 95.93%, respectively. The results are beneficial to the large-scale extraction of anthocyanins from black rice. Practical Applications Due to the growing demand for natural pigments, the natural anthocyanins have drawn great attentions. However, the immaturity of the large-scale purification procedures has limited the application of anthocyanins in functional food and pharmaceutical industries. Since the black rice is the rich source of anthocyanins, the large-scale water extraction and macroporous absorbent resins purification parameters were systematically investigated in the current study. About 0.86 g anthocyanins could be obtained from per 100 g black rice based on the large-scale method, which has a good recovery, and the purity could reach 95.93%. Meanwhile, the whole procedure was green and non-pollution as the primary solvents were water and ethanol. Furthermore, the results also would be conducive to the processing and utilization of black rice.

A novel rapid and continuous procedure for large-scale purification of magnetosomes from Magnetospirillum gryphiswaldense

A new rapid and continuous procedure was developed for purifying magnetosomes from Magnetospirillum gryphiswaldense MSR-1 cells on a large scale. The procedure included these steps: disruption of cells with a high-pressure homogeniser, isolation of magnetosomes with a continuous magnetism isolation system accompanied by low-power ultrasonication and urea treatment, removal of adsorbed and surface proteins with proteinase K, removal of nucleic acids with electro-elution, and replacement of the PBS buffer with distilled water by a magnetically stirred system. The purified magnetosomes were stored at -20 °C after lyophilized and treated with γ-rays. The time required for purification was reduced from 20-30 to 2-5 days. Evaluation of the purity of the resulting magnetosomes was carried out with SDS-PAGE, PCR, and Fourier-transform infrared spectroscopy. The overall data suggest that the method presented here is a simple, rapid, continuous, and highly efficient procedure for large-scale purification of magnetosomes.

Single Channel Sensing of dsDNA using the Membrane- Adapted Nanopore of phi29 DNA Packaging Motor

The bacteriophage phi29 DNA-packaging motor, geared by six pRNA molecules, contains a truncated cone shape connector channel that is 3.6nm in diameter at its narrow end and 6nm in diameter at its wide end. This channel allows dsDNA to enter and exit the virus procapsid during virus maturation and infection, respectively. We modified the genes that code for the core of the phi29 DNA packaging motor in order to change the amino acid sequence of its protein for membrane incorporation. The modified connector was reconstituted into liposomes and fused into a planar lipid bilayer membrane. Distinctive current jumps were found for each connector insertion. The conductance of each connector channel was measured and found to be uniform (4.8nS in 1M KCl). The membrane embedded connector channel was found to be able to translocate dsDNA. The translocations were recorded as blockage events of the current. The blockages were equal for each of the individual channels, generating a clean, homogenous and uniform signal representing DNA translocation.The connector channel is larger than the previously studied ion channels, which could only let ssDNA pass. The robust property of the connector in ion and dsDNA tranlocation has extensive potentials in microelectromechanical sensing, microreactors, gene delivery, drug loading and DNA sequencing. Single molecule and low concentration sensing can be achieved using this membrane embedded connector system. Additionally, the available crystal structure of the connector protein makes it easy to modify the channel for specific applications and the established large scale purification procedure of the connector will facilitate its practice.

Purification of messenger ribonucleoprotein particles from rabbit reticulocytes by zonal centrifugation in metrizamide.

A large-scale purification procedure for messenger ribonucleoprotein (mRNP) particles from rabbit reticulocyte polysomes is described. The mRNP particles were dissociated from polysomes by treatment with urea and separated by differential centrifugation under conditions of high ionic strength. Zonal centrifugation in a metrizamide buoyant density gradient was the final purification step. One major class of mRNA particle was observed. The RNA was defined as mRNA by polyacrylamide gel electrophoresis and by globin production in a cell-free protein-synthesizing system. The twenty-three different proteins associated with the particle were a discrete set of proteins, which ranged in molecular weight from 175,000 to 23,500. The relative amount of each peptide in the particle was determined from a gel scan of the stained protein by computer simulation. None of the polypeptides comigrated with proteins from the 40-S and 60-S ribosomal subunits when analyzed by two-dimensional polyacrylamide gel electrophoresis.

Establishment of a Large-Scale Purification Procedure for Purified Recombinant Bovine Interferon-.TAU. Produced by a Silkworm-Baculovirus Gene Expression System

We developed a procedure for the large-scale purification of bovine interferon-tau (boIFN-tau) by means of a silkworm-baculovirus gene expression system. Recombinant boIFN-tau (rboIFN-tau) was efficiently produced in the silkworm infected with boIFN-tau cDNA recombinant baculovirus and accumulated in the haemolymph. To establish a purification method suitable for mass production, we tried three crude purification methods, namely, an acidification and neutralization treatment (ANT), silica gel column chromatography (SGCC), and Blue sepharose column chromatography (BSCC) with a combination of Q-sepharose (QSC) and chelating sepharose column chromatographies (CSCC). As a result, the acidification and neutralization treatment was found to be the most efficient and cost effective. With this combination, we obtained 91% pure products. To confirm the applicability of the procedure for mass production, we inoculated 100 silkworms with the recombinant virus, and recovered about 4.55 mg (1.26 x 10(8)U/mg) of 91% pure rboIFN-tau by means of a combination of the ANT, followed by QSC and CSCC.

Development of a large-scale HPLC-based purification for the urease from Staphylococcus leei and determination of subunit structure

Coagulase-positive Staphylococcus species, related to but distinct from the genetic homology group containing Staphylococcus cohnii, Staphylococcus xylosus, and Staphylococcus saphrophyticus, were isolated from biopsy material obtained from a cluster of patients in Korea suffering from gastritis. The prototype isolate, Staphylococcus leei, has high urease activity that is similar with respect to a low K m value and acid resistance of the urease found in the stomach adapted pathogen, Helicobacter pylori. S. leei is remarkably resistant to lysis and only a small fraction of the cells are broken using sonication, a French press, Niro homogenizer, or a Gaulin mill. In the present report, we describe an efficient cell lysis procedure for S. leei using three passes through a Dynomill with 0.5 mm glass beads that results in lysis of more than 95% of the cells. We also developed an efficient and large-scale purification procedure for the S. leei urease using a BioCAD HPLC Workstation using Q-Sepharose, Poros HP2, Sephacryl S-300, and hydroxyapatite chromatography. The urease of S. leei was purified 98-fold to a specific activity of 731 U/mg. The urease protein is composed of three subunits, α (65 kDa), β (21 kDa), and γ (12 kDa), and in situ enzyme assay and molecular sieve chromatography indicate that multiple high molecular weight forms are present, including an apparent pentamer of 1:1:1 αβγ-heterotrimers of 480 kDa. This purification procedure was used to purify 16 mg of enzyme from 120-liters of cell culture. This improved lysis and purification procedure will make it possible to obtain sufficient quantities of urease for use as an antigen in ELISA assays to carry out studies to determine the incidence and demographic prevalence of gastritis due to S. leei.

Flexibility revealed by the 1.85 A crystal structure of the beta sliding-clamp subunit of Escherichia coli DNA polymerase III.

The beta subunit of the Escherichia coli replicative DNA polymerase III holoenzyme is the sliding clamp that interacts with the alpha (polymerase) subunit to maintain the high processivity of the enzyme. The beta protein is a ring-shaped dimer of 40.6 kDa subunits whose structure has previously been determined at a resolution of 2.5 A [Kong et al. (1992), Cell, 69, 425-437]. Here, the construction of a new plasmid that directs overproduction of beta to very high levels and a simple procedure for large-scale purification of the protein are described. Crystals grown under slightly modified conditions diffracted to beyond 1.9 A at 100 K at a synchrotron source. The structure of the beta dimer solved at 1.85 A resolution shows some differences from that reported previously. In particular, it was possible at this resolution to identify residues that differed in position between the two subunits in the unit cell; side chains of these and some other residues were found to occupy alternate conformations. This suggests that these residues are likely to be relatively mobile in solution. Some implications of this flexibility for the function of beta are discussed.

Large-Scale Expression and Purification of High-Molecular-Weight Glutenin Subunits

The high-molecular-weight glutenin subunits (HMW-GSs) are considered to be one of the most important components of wheat gluten, contributing to the unique viscoelastic properties of wheat dough. The HMW-GSs are highly homologous in sequence and structure and a mixture of subunits is usually present in wheat flours. Consequently, it is difficult to purify these proteins separately in appreciable amounts. Expression in heterologous systems provides a clear opportunity to produce large amounts of single HMW-GS proteins, amounts (up to 100 mg) which are required for in vitro analysis of these proteins. However, since the first expression studies of HMW-GSs, over 10 years ago, this technology has not been widely utilized. Previous studies have been analytical or small scale (5–100 ml) and in most cases only partial purity was obtained. In the present paper, we describe in detail the expression of the HMW-GSs Glu1-Dx2, Dx5, Dy10, and Dy12 for the first time on a large scale, producing up to 100 mg of target protein from a 2-liter bacterial culture, using a Biostat fermenter. Our results include optimization of expression conditions to increase yield and stability of proteins. Results also include localization, differences between x- and y-type expression and small-scale versus large-scale expression. We also developed a large-scale purification procedure. The bacterially expressed proteins have the same molecular weight on SDS-PAGE and the same retention times on RP-HPLC as their native counterparts extracted from flour. Functionality tests, on the bacterially produced proteins, have shown a clear correlation with the equivalent native proteins from flour. These results provide a clear opportunity to produce protein in amounts necessary for more detailed studies of the structure and function of the HMW-GSs and glutenin polymers on dough development and quality.

Rapid Two-Step Procedure for Large-Scale Purification of Pediocin-Like Bacteriocins and Other Cationic Antimicrobial Peptides from Complex Culture Medium

A rapid and simple two-step procedure suitable for both small- and large-scale purification of pediocin-like bacteriocins and other cationic peptides has been developed. In the first step, the bacterial culture was applied directly on a cation-exchange column (1-ml cation exchanger per 100-ml cell culture). Bacteria and anionic compounds passed through the column, and cationic bacteriocins were subsequently eluted with 1 M NaCl. In the second step, the bacteriocin fraction was applied on a low-pressure, reverse-phase column and the bacteriocins were detected as major optical density peaks upon elution with propanol. More than 80% of the activity that was initially in the culture supernatant was recovered in both purification steps, and the final bacteriocin preparation was more than 90% pure as judged by analytical reverse-phase chromatography and capillary electrophoresis.

Purification des nanotubes de carbone monofeuillets

The electric arc method allows to produce large quantities (a few grams per day) of single walled carbon nanotubes (SWNT) which coexist with metallic particles and other forms of carbon: amorphous carbon, graphite… The large scale purification procedure presented in this note allows to get grams per week of SWNT purified at more than 90 %. The procedure used consists in a nitric acid treatment of the raw material, followed by a cross flow filtration step and a heat treatment at 1600 °C under nitrogen atmosphere. The efficiency of the method is characterized by electron microscopy (SEM and HRTEM) and discussed step by step.

Spectroscopic investigations on the water-soluble fragment of the Rieske [2Fe2S] protein from Paracoccus denitrificans

A large-scale purification procedure has been developed to isolate the membrane-bound bc 1 complex of Paracoccus denitrificans. From the purified complex, a water-soluble fragment has been isolated. The fragment comprising residues 50–190 contains the Rieske [2Fe2S] cluster and has an optical spectrum which is typical of such a cluster. The EPR spectrum of the cluster in the bc 1 complex differs slightly fr the spectrum of the water-soluble fragment. Differences in the g values and linewidths of the resonances can be attributed to the absence of bound quinone in the water-soluble fragment. Signals from the cysteines and histidines that coordinate to the [2Fe2S] cluster could be identified in the 1H NMR spectrum of the fragment in solution.

A rapid purification protocol for the mitogen-activated p70 S6 kinase.

Reentry into the cell cycle from quiescence is stimulated by mitogens and supported by increased levels of protein synthesis. Initiation of polypeptide synthesis is the step most often subject to regulation. This is controlled by phosphorylation of a number of initiation factors and the single 40S ribosomal protein S6. The kinase responsible for S6 phosphorylation is p70(S6k). In the past, the apparent low cellular abundance of p70(S6k) resulted in the development of laborious large-scale purification procedures. Here a rapid and low-cost protocol which yields p70(S6k) purified to near homogeneity and with high specific activity is described. With respect to previous strategies, this novel scheme takes advantage of recently improved ion-exchange media in the first and second purification steps, thus allowing the effective removal of bulk proteins and the major Ser/Thr phosphatases. The third step of purification consists of a single affinity chromatography column in which the ligand is a 20-residue peptide containing the structural motif required for recognition and binding by p70(S6k). This novel protocol allows the rapid purification of large amounts of p70(S6k) and will facilitate the screening of libraries of natural or synthetic compounds aimed at the identification of p70(S6k) inhibitory molecules.

17] Purification of yeast mitochondrial chaperonin 60 and co-chaperonin 10

Publisher Summary The chapter presents a study on the purification of yeast mitochondrial chaperonin 60 and co-chaperonin 10. The chapter describes a large-scale purification procedure of S. cerevisiae hsp60 expressed in Pichia pastoris. Yeast cpnl0 was isolated by taking advantage of the fact that bacterial GroEL forms a functional complex with co-chaperonins from mitochondria or chloroplasts. Chaperonin system is not involved in the folding of all mitochondrial proteins. Assaying the interaction of purified hsp60 and cpnl 0 with authentic substrate proteins should give insight into the specificity of the mitochondrial chaperonin system. In addition, a comparison of the bacterial GroEL/ES system with the eukaryotic hsp60/cpnl0 system may help to elucidate the chaperonin reaction cycle. The chapter describes the procedure for purification of recombinant heat shock protein 60, purification of recombinant authentic and hexahistidine-tagged chaperonin 10, and also mentions the characterization of purified yeast chaperonins.

Characterization of Di-myo-Inositol-1,1(prm1)-Phosphate in the Hyperthermophilic Bacterium Thermotoga maritima

Di-myo-inositol-1,1(prm1)-phosphate (DIP) is present at a significant concentration ((symbl)160 nmol/mg of protein) in the cytoplasm of the hyperthermophilic bacterium Thermotoga maritima. The concentration of DIP was independent of the pH of the growth medium or the cell growth phase but increased with increasing concentrations of NaCl in the growth medium, reaching a maximum ((symbl)450 nmol/mg of protein) at 0.4 to 0.6 M NaCl. A large-scale purification procedure for DIP which yields approximately 18 g of DIP per kg of cells (wet weight) is described. Purified DIP was stable at 90(deg)C for at least 5 h. The presence of DIP (50 mM) did not increase the stability at 90(deg)C of pure forms of the hydrogenase or pyruvate ferredoxin oxidoreductase of T. maritima, suggesting that DIP is not a general thermoprotectant.

A simple procedure for large-scale purification of 9-cis beta-carotene from Dunaliella bardawil.

For experiments designed to obtain reliable data on the metabolic pathway and biological function of 9-cis beta-carotene, sufficient quantity of this substance in pure form is needed. For this purpose, we decided to purify 9-cis beta-carotene from the dry powder of the alga Dunaliella bardawil. By use of both silica gel and ODS open column chromatography and high-performance liquid chromatography (HPLC) on an ODS column and by treatment of the sample with ethanol, we could obtain this carotenoid as fine needle-shaped orange crystals. They were found to be highly pure as judged by analytical HPLC, absorption spectrum, and nuclear magnetic resonance analysis.

Recombinant human liver medium-chain acyl-CoA dehydrogenase: purification, characterization, and the mechanism of interactions with functionally diverse C8-CoA molecules.

We offer a large scale purification procedure for the recombinant human liver medium-chain acyl-CoA dehydrogenase (HMCAD). This procedure routinely yield 100-150 mg of homogeneous preparation of the enzyme from 80 L of the Escherichia coli host cells. A comparative investigation of kinetic properties of the human liver and pig kidney enzymes revealed that, except for a few minor differences, both of these enzymes are nearly identical. We undertook detailed kinetic and thermodynamic investigations for the interaction of HMCAD-FAD with three C8-CoA molecules (viz., octanoyl-CoA, 2-octenoyl-CoA, and 2-octynoyl-CoA), which differ with respect to the extent of unsaturation of the alpha-beta carbon center; octanoyl-CoA and 2-octenoyl-CoA serve as the substrate and product of the enzyme, respectively, whereas 2-octynoyl-CoA is known to inactivate the enzyme. Our experimental results demonstrate that all three C8-CoA molecules first interact with HMCAD-FAD to form corresponding Michaelis complexes, followed by two subsequent isomerization reactions. The latter accompany either subtle changes in the electronic structures of the individual components (in case of 2-octenoyl-CoA and 2-octynoyl-CoA ligands), or a near-complete reduction of the enzyme-bound flavin (in case of octanoyl-CoA). The rate and equilibrium constants intrinsic to the above microscopic steps exhibit marked similarity with different C8-CoA molecules. However, the electronic structural changes accompanying the 2-octynoyl-CoA-dependent inactivation of enzyme is 3-4 orders of magnitude slower than the above isomerization reactions. Hence, the octanoyl-CoA-dependent reductive half-reaction and the 2-octynoyl-CoA-dependent covalent modification of the enzyme occur during entirely different microscopic steps. Arguments are presented that the origin of the above difference lies in the protein conformation-dependent orientation of Glu-376 in the vicinity of the C8-CoA binding site.

A Modified Form of the Cytochrome P450scc Precursor: A New Approach in Studies on Protein Import into Mitochondria

Recombinant DNA was constructed providing hypersynthesis of a hybrid protein with a MRGSH6GIR sequence preceding the NH2-terminus of the bovine cytochrome P450scc precursor (6His-pP450scc) in Escherichia coli cells. A large-scale procedure for isolation and purification of this protein was elaborated. 6His-pP450scc was imported into isolated rat liver mitochondria and processed to the mature-sized form. As a similar procedure can be applied to other proteins the results of this work offer new opportunities in studies of protein import into mitochondria.

Immunoaffinity Purification of Active Rat Recombinant 1,25-Dihydroxyvitamin D 3 Receptor

We have developed a large-scale immunoaffinity purification procedure for the recombinant vitamin D receptor. The purified receptor is homogenous, and is bound by 1,25-dihydroxyvitamin D 3 with a K d of 5 × 10 −10 M. The isolated receptor binds to the osteocalcin vitamin D response element in the presence of porcine intestinal nuclear extract stripped of endogenous vitamin D receptor as well. However, the binding of D 3 and the vitamin D 3 response element does not completely assure a native conformation of the protein. The availability of large quantities of highly purified active vitamin D receptor makes possible detailed structural analysis.

Molecular identification of the ten subunits of cytochrome-c reductase from potato mitochondria.

Cytochrome-c reductase (EC 1.10.2.2.) from Solanum tuberosum L. comprises ten subunits with apparent molecular sizes of 55, 53, 51, 35, 33, 25, 14, 12, 11 and 10 kDa on 14% SDS-PAGE. The identity of the subunits was analysed by direct amino-acid sequencing via cyclic Edman degradation. A large-scale purification procedure for the enzyme complex based on affinity chromatography and gelfiltraton is described. All subunits were enzymatically fragmented and the generated peptides were separated by reverse-phase HPLC. Complete or partial sequence determination of 33 peptides comprising a total of nearly 500 amino acids showed, that cytochrome-c reductase from potato contains three respiratory proteins (cytochrome b, cytochrome c1, and the "Rieske" iron-sulfur protein), four small proteins with molecular sizes below 15 kDa (so-called Q-binding, hinge, cytochrome-c1-linked and core-linked proteins) and three proteins in the 50-kDa range which show similarity to members of the core/PEP/MPP protein family (core/processing enhancing protein/mitochondrial processing peptidase). In fact these subunits show highest sequence identity either to MPP or PEP, which is in line with earlier findings, that isolated cytochrome-c reductase from potato exhibits processing activity towards mitochondrial precursor proteins.