Five-step Purification Research Articles

Expression and purification of snustorr snarlik protein from Plutella xylostella

Snustorr snarlik (Snsl) is a type of extracellular protein essential for insect cuticle formation and insect survival, but is absent in mammals, making it a potential selective target for pest control. Here, we successfully expressed and purified the Snsl protein of Plutella xylostella in Escherichia coli. Two truncated forms of Snsl protein, Snsl 16–119 and Snsl 16–159, were expressed as a maltose-binding protein (MBP) fusion protein and purified to a purity above 90% after a five-step purification protocol. Snsl 16–119, forming stable monomer in solution, was crystallized, and the crystal was diffracted to a resolution of ∼10 Å. Snsl 16–159, forming an equilibrium between monomer and octamer in solution, was shown to form rod-shaped particles on negative staining electron-microscopy images. Our results lay a foundation for the determination of the structure of Snsl, which would improve our understanding of the molecular mechanism of cuticle formation and related pesticide resistance and provide a template for structure-based insecticide design.

Molecular design and downstream processing of turoctocog alfa (NovoEight), a B-domain truncated factor VIII molecule.

Turoctocog alfa (NovoEight) is a third-generation recombinant factor VIII (rFVIII) with a truncated B-domain that is manufactured in Chinese hamster ovary cells. No human or animal-derived materials are used in the process. The aim of this study is to describe the molecular design and purification process for turoctocog alfa. A five-step purification process is applied to turoctocog alfa: protein capture on mixed-mode resin; immunoaffinity chromatography using a unique, recombinantly produced anti-FVIII mAb; anion exchange chromatography; nanofiltration and size exclusion chromatography. This process enabled reduction of impurities such as host cell proteins (HCPs) and high molecular weight proteins (HMWPs) to a very low level. The immunoaffinity step is very important for the removal of FVIII-related degradation products. Manufacturing scale data shown in this article confirmed the robustness of the purification process and a reliable and consistent reduction of the impurities. The contribution of each step to the final product purity is described and shown for three manufacturing batches. Turoctocog alfa, a third-generation B-domain truncated rFVIII product is manufactured in Chinese hamster ovary cells without the use of animal or human-derived proteins. The five-step purification process results in a homogenous, highly purified rFVIII product.

A new recombinant factor VIII: from genetics to clinical use.

Advances in recombinant technology and knowledge about coagulation factor VIII (FVIII) are building a platform for new therapeutic options in patients with hemophilia A. The development of turoctocog alfa, a novel, high-purity, third-generation, B-domain truncated recombinant FVIII, has been produced and formulated without the use of animal-derived or human serum-derived components, in the wake of understanding of the new biochemical characteristics of FVIII, namely its protein structure, and glycosylation and sulfating patterns. Culture conditions and a five-step purification process have been developed to optimize the safety of turoctocog alfa. The results of two pilot clinical trials using turoctocog alfa confirmed high safety levels, with no patient developing inhibitors during the period of observation. The purpose of this review is to describe briefly the molecular and biological properties of turoctocog alfa, together with details of its clinical development, with emphasis on the needs of patients with hemophilia A.

Purification and characterization of a cold-adapted pullulanase from a psychrophilic bacterial isolate.

There is a considerable potential of cold-active biocatalysts for versatile industrial applications. A psychrophilic bacterial strain, Shewanella arctica 40-3, has been isolated from arctic sea ice and was shown to exhibit pullulan-degrading activity. Purification of a monomeric, 150-kDa pullulanase was achieved using a five-step purification approach. The native enzyme was purified 50.0-fold to a final specific activity of 3.0 U/mg. The enzyme was active at a broad range of temperature (10-50 °C) and pH (5-9). Optimal activity was determined at 45 °C and pH 7. The presence of various metal ions is tolerated by the pullulanase, while detergents resulted in decreased activity. Complete conversion of pullulan to maltotriose as the sole product and N-terminal amino acid sequence indicated that the enzyme is a type-I pullulanase and belongs to rarely characterized pullulan-degrading enzymes from psychrophiles.

Crystallization and preliminary crystallographic analysis of human muscle phosphofructokinase, the main regulator of glycolysis

Whereas the three-dimensional structure and the structural basis of the allosteric regulation of prokaryotic 6-phosphofructokinases (Pfks) have been studied in great detail, knowledge of the molecular basis of the allosteric behaviour of the far more complex mammalian Pfks is still very limited. The human muscle isozyme was expressed heterologously in yeast cells and purified using a five-step purification protocol. Protein crystals suitable for diffraction experiments were obtained by the vapour-diffusion method. The crystals belonged to space group P6222 and diffracted to 6.0 Å resolution. The 3.2 Å resolution structure of rabbit muscle Pfk (rmPfk) was placed into the asymmetric unit and optimized by rigid-body and group B-factor refinement. Interestingly, the tetrameric enzyme dissociated into a dimer, similar to the situation observed in the structure of rmPfk.

Biochemical characterization of aspartate aminotransferase allozymes from common wheat

AbstractSix allozymes of aspartate aminotransferase (AAT, EC 2.6.1.1): three plastidial (AAT-2 zone) and three cytosolic (AAT-3 zone) were isolated from common wheat (Triticum aestivum) seedlings and highly purified by a five-step purification procedure. The identity of the studied proteins was confirmed by mass spectrometry. The molecular weight of AAT allozymes determined by gel filtration was 72.4±3.6 kDa. The molecular weights of plastidial and cytosolic allozymes estimated by SDS-PAGE were 45.3 and 43.7 kDa, respectively. The apparent Michaelis constant (K m) values determined for four substrates appeared to be very similar for each allozyme. The values of the turnover number (k cat) and the k cat/K m ratio calculated for allozymes with L-aspartate as a leading substrate were in the range of 88.5–103.8 s−1/10,412–10,795 s−1 M−1 for AAT-2 zone and 4.6–7.0 s−1/527–700 s−1 M−1 for AAT-3 zone. These results clearly demonstrated much higher catalytic efficiency of AAT-2 allozymes. Therefore, partial sequences of cDNA encoding AATs from different zones were obtained using the RT-PCR technique. Comparison of the AAT-2 and AAT-3 amino acid sequences from active site regions revealed five non-conservative substitutions, which impact on the observed differences in the isozymes catalytic efficiency is discussed.

Biochemical characterisation of prolyl aminopeptidase from shoots of triticale seedlings and its activity changes in response to suboptimal growth conditions

Prolyl aminopeptidase (PAP) was isolated from the shoots of three-day-old triticale seedlings and was purified using a five-step purification procedure (acid precipitation, gel filtration, anion-exchange chromatography, hydrophobic chromatography and rechromatography). The enzyme was purified 460-fold with a recovery of 6%. Prolyl aminopeptidase appears to be a tetramer consisting of four subunits, each with a molecular weight of approximately 54kDa. Its pH and temperature optimum are pH 7.5 and 37°C, respectively. The enzyme prefers substrates with Pro and Hyp at the N-terminus, but is also capable of hydrolysing β-naphthylamides (β-NA) of Ala, Phe, and Leu. The K(m) value of PAP against Pro-β-NA was the lowest among the substrates tested and it was 1.47×10(-5)M. The activity of PAP was not inhibited by EDTA, 1,10-phenantroline, or pepstatin A. The most effective inhibitors were DFP, Pefabloc, and PMSF, which are serine protease inhibitors. However, significant inhibition was also observed in the presence of E-64, which modifies sulfhydryl groups. A significant increase of the aminopeptidase activity against Pro-β-NA was observed in shoots of triticale plants grown under salinity, drought stress, and in the presence of cadmium and aluminium ions in the nutrient solution.

Purification and characterization of acetophenone reductase with excellent enantioselectivity from Geotrichum candidum NBRC 4597

NADH-dependent enzyme reducing acetophenone derivatives with high stereoselectivities and wide substrate specificities from Geotrichum candidum NBRC 4597 was isolated, purified, characterized, and used for asymmetric synthesis. Through five-step purification including ammonium sulfate fractionation and a series of chromatographies, the enzyme was purified about 150-fold with a yield of 5.6%. The active enzyme has a molecular mass of 73 kDa determined by gel filtration chromatography, and the SDS-PAGE result reveals that the molecular size of the subunit is 36 kDa. These results indicate that the enzyme consists of a homodimer of a 36 kDa subunit. The acetophenone reductase exhibited the highest activity at 50 degrees C and optimal pH at 5.5. The enzyme was the most stable at 40 degrees C. No metal ions considerably activated the enzyme, and such metal ions as Cu2+, Cd2+, and Zn2+ strongly inhibited the activity of the enzyme. The Vmax and the apparent Km value of the reductase were 77.0 micromol/min per milligram of protein and 0.296 mM for acetophenone, respectively. The N-terminal and internal amino acid sequences were determined by peptide sequencer. Furthermore, the purified enzyme was used for asymmetric reduction of acetophenone, resulting in the formation of corresponding (S)-alcohol with 99% ee.

Purification and identification of an angiotensin I-converting enzyme inhibitory peptide from fermented soybean extract

Angiotensin I-converting enzyme (ACE), a dipeptidyl carboxypeptidase, plays an important physiological role in regulating blood pressure. ACE-inhibitory peptides derived from food proteins have potential pharmaceutical and human health uses. In this study, we prepared a fermented soybean extract (FSE) through a rapid fermentation at an elevated temperature to accelerate proteolytic hydrolysis and described purification procedures to discover potent ACE-inhibitory peptides from FSE. After 3 days of aging, FSE exhibited ACE-inhibitory activity with an IC 50 value of 1.46 mg/mL. Purification of novel ACE-inhibitory peptides was carried out using ultrafiltration and consecutive chromatographic methods. A novel ACE-inhibitory peptide, with 66-fold increase in ACE-inhibitory activity compared to that of FSE, was isolated from FSE through a five-step purification procedure. The amino acid sequence of the purified ACE-inhibitory peptides was determined to be Leu-Val-Gln-Gly-Ser by Edman degradation method, and its IC 50 value was 22 μg/mL (43.7 μM).

Purification and identification of adipogenesis inhibitory peptide from black soybean protein hydrolysate

Adipogenesis inhibitory peptide was isolated and identified from black soybean ( Rhynchosia volubilis Lour .) hydrolysate. An adipogenesis inhibitor was purified using consecutive methods including: ultrafiltration (MWCO; 3 and 10 kDa), gel filtration chromatography (Superdex Peptide 10/300 GL column), and reverse-phase high-performance liquid chromatography (μBondapak™ C 18 column). Also, the adipogenesis inhibition effect of the purified peptide was measured by observation of droplet of 3T3-L1 adipocyte by Oil Red O staining in the highest active fraction in each step. The peptide was shown to inhibit the differentiation of the 3T3-L1 pre-adipocyte, which was confirmed by morphological study. The adipogenesis inhibitory peptide was purified 71.43-fold from black soybean hydrolysate throughout a five-step purification procedure. The adipogenesis inhibitor was identified to be a tripeptide, Ile-Gln-Asn, having an IC 50 value of 0.014 mg protein/ml. Furthermore, the synthetic tripeptide (Ile-Gln-Asn) exhibited the similar adipogenesis effects to the purified peptide. Thus, these results showed the potential anti-obesity effect of the purified peptide through control of adiposity.

Purification from normal human plasma and biochemical characterization of a ribonuclease specific for poly(C) and poly(U)

A new specific ribonuclease from normal human plasma has been purified to homogeneity, following a five-step purification protocol that included DEAE–Sepharose, CM–Sepharose, and Heparin–Sepharose chromatographies. The purified enzyme was found to be glycosylated and appeared as a single 25-kDa band on a SDS polyacrylamide gel. This RNase is poly(C) preferential, degrading poly(U) at a lower rate. Activity of this RNase toward cleavage of native substrates such as ribosomal RNA was also detected. The human plasma ribonuclease is a thermolabile molecule, exhibiting maximum activity at pH 6.5. Comparison between other known plasma RNases and the human plasma ribonuclease described here indicated a variety of differences in their biochemical and catalytic properties.

Isolation, Purification, and Properties of Malate Dehydrogenase from Sulfur Bacterium Beggiatoa leptomitiformis

Malate dehydrogenase (E.C. 1.1.1.37) from the bacterium Beggiatoa leptomitiformis was isolated and purified 123-fold using a five-step purification procedure including the enzyme extraction, ammonium sulfate protein fractionation, gel filtration, ion exchange chromatography, and gel chromatography. The enzyme was homogenous according to the electrophoresis data; its activity was 20.43 U/mg protein. This malate dehydrogenase is a homotetramer (Mr = 172 kDa). The catalytic and thermodynamic properties, as well as the analysis of the published data suggest that the tetrameric structure of the enzyme allows it to participate in constructive metabolism supplying the cell with organic acids as a source of carbon.

A newly-detected reductase from Rauvolfia closes a gap in the biosynthesis of the antiarrhythmic alkaloid ajmaline.

A new enzyme, 1,2-dihydrovomilenine reductase (E.C. 1.3.1), has been detected in Rauvolfia cell suspension cultures. The enzyme specifically converts 2beta( R)-1,2-dihydrovomilenine through an NADPH-dependent reaction into 17-O-acetylnorajmaline, a close biosynthetic precursor of the antiarrhythmic alkaloid ajmaline from Rauvolfia. A five-step purification procedure using SOURCE 30Q chromatography, hydroxyapatite chromatography, 2',5'-ADP Sepharose 4B affinity chromatography and ion exchange chromatography on DEAE Sepharose and Mono Q delivered an approximately 200-fold enriched enzyme in a yield of approximately 6%. SDS-PAGE showed an M r for the enzyme of approximately 48 kDa. Optimum pH and optimum temperature of the reductase were at pH 6.0 and 37 degrees C. The enzyme shows a limited distribution in cell cultures expressing ajmaline biosynthesis, and is obviously highly specific for the ajmaline pathway.

Induction of aconitate hydratase in hepatocytes of starving rats.

Induction of the activity of aconitate hydratase (AH) was observed in rat hepatocytes under the conditions of food deprivation. The increase in AH activity after 4 days of starvation in the studied tissues was from 0.57 to 2.05 U/g crude liver weight. The induction of aconitase was associated both with the cytoplasmic and mitochondrial AH isoforms. The activities of cytosolic and mitochondrial AH isoforms in starving animals consisted of 83 and 17% of the total activity, respectively. The cytoplasmic and mitochondrial isoforms of the enzyme with specific activities 11.1 and 6.13 U/mg protein, respectively, were obtained by a five-step purification procedure that included fractionation with ammonium sulfate, ion-exchanging chromatography on DEAE-Toyopearl and gel filtration. The purified preparations of these AH isoforms were electrophoretically homogenous. The molecular weights of these isoforms were estimated and several kinetic and regulatory properties were studied.

Crystallization and preliminary X-ray diffraction analysis of Thermus thermophilus prolyl-tRNA synthetase.

Prolyl-tRNA synthetase from Thermus thermophilus (ProRSTT) was purified to homogeneity using a five-step purification procedure and was crystallized using ethylene glycol as a precipitant. Crystals of ProRSTT belong to the space group P2(1)2(1)2, with unit-cell parameters a = 132, b = 191, c = 125 A, have two homodimers per asymmetric unit and diffract to 2.4 A resolution. A complete native data set to 2.43 A resolution has been collected and a data set from ProRSTT in complex with proline has been collected to 2.9 A resolution.

Enzymatic detoxification of eutypine, a toxin from Eutypa lata , by Vitis vinifera cells: partial purification of an NADPH-dependent aldehyde reductase

Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzaldehyde, is a toxin produced by Eutypa lata (Pers.: Fr.) Tul., the causal agent of dying arm disease of Vitis vinifera L. (grapevine). Previously, we have shown that eutypine is involved in the development of disease symptoms. In the present study, the effects of V. vinifera cell-suspension cultures on the biological activity of the toxin were investigated. Eutypine was converted by grapevine tissues into a single compound, identified by mass spectrometry and nuclear magnetic resonance as 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzyl alcohol, designated eutypinol. This compound was found to be non-toxic for grapevine tissues. Unlike eutypine, eutypinol failed to affect the oxidation rate or membrane potential of isolated mitochondria. In grapevine cells, reduction of eutypine into the corresponding alcohol is an NADPH-dependent enzymatic reaction. An enzyme which reduced eutypine was partially purified, over 1000-fold, using a five-step purification procedure. By gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein was found to have a molecular mass of 54–56 kDa. The enzyme exhibited an apparent Km for eutypine of 44 μM, and was active between pH 6.8 and 7.5 with a maximum at pH 7.0. The eutypine reductase activity was improved by Mn2+ and Mg2+ and inhibited by disulfiram and p-hydroxymercuribenzoate. The possible role of the eutypine-detoxification mechanism in the defense reactions of V. vinifera cells is discussed.

23] Bovine mitochondrial initiation and elongation factors

The procedures summarized above provide nearly homogeneous preparations of IF-2mt, EF-Tu. Tsmt, and EF-Gmt. The scheme developed for IF-2mr leads to a 24,000-fold purification of this factor with a 26% recovery of activity. Analysis by SDS-polyacrylamide gel electrophoresis and gel filtration chromatography indicates that this factor functions as a monomer with a molecular weight of about 85,000. The scheme developed EF-Tu.Tsmt provides a 10,000-fold purification with an overall yield of about 10%. The EF-Tumt component in this complex has a molecular weight of about 46,000, whereas EF-Tsmt has a molecular weight of about 32,000 on SDS-polyacrylamide gel electrophoresis. The EF-Tu. Tsmt complex is tightly associated and appears to have a native molecular weight of about 70,000. The five-step purification procedure outlined above for EF-Gmt results in a 14,000-fold purification of EF-Gmt with a 2-5% recovery of activity. Analysis by SDS-polyacrylamide gel electrophoresis and gel filtration chromatography indicates that EF-Gmt functions as a monomeric protein with an apparent molecular weight of about 80,000.

Dehalogenation of 4-chlorobenzoate

Pseudomonas sp. CBS3 is capable of growing with 4-chlorobenzoate as sole source of carbon and energy. The removal of the chlorine of 4-chlorobenzoate is performed in the first degradation step by an enzyme system consisting of three proteins. A 4-halobenzoate-coenzyme A ligase activates 4-chlorobenzoate in a coenzyme A, ATP and Mg2+ dependent reaction to 4-chlorobenzoyl-coenzyme A. This thioester intermediate is dehalogenated by the 4-chlorobenzoyl-coenzyme A dehalogenase. Finally coenzyme A is split off by a 4-hydroxybenzoyl-CoA thioesterase to form 4-hydroxybenzoate. The involved 4-chlorobenzoyl-coenzyme A dehalogenase was purified to apparent homogeneity by a five-step purification procedure. The native enzyme had an apparent molecular mass of 120,000 and was composed of four identical polypeptide subunits of 31 kDa. The enzyme displayed an isoelectric point of 6.7. The maximal initial rate of catalysis was achieved at pH 10 at 60 degrees C. The apparent Km value for 4-chlorobenzoyl-coenzyme A was 2.4-2.7 microM. Vmax was 1.1 x 10(-7) M sec-1 (2.2 mumol min-1 mg-1 of protein). The NH2-terminal amino acid sequence was determined. All 4-halobenzoyl-coenzyme A thioesters, except 4-fluorobenzoyl-coenzyme A, were dehalogenated by the 4-chlorobenzoyl-CoA dehalogenase.

Purification of SEF1 proteins binding to transcriptional enhancer elements active in T lymphocytes.

Binding sites for SL3-3 enhancer factor 1 (SEF1) are important for the transcriptional activity in T lymphocytes and the tumorigenicity of SL3-3 murine leukemia virus. SEF1 is also implicated in the activity of many other leukemia, lymphoma, and sarcoma virus enhancers, and enhancers of genes for T cell receptor-CD3 subunits. We have purified several proteins binding to SEF1 sites from bovine thymus using a five-step purification procedure. The proteins migrated as 19 distinct bands representing molecular masses from 23 kDa to about 200 kDa in SDS-polyacrylamide gel electrophoresis. Ten DNA binding proteins, with molecular masses between 23 and 67 kDa, could be isolated after separation by SDS-polyacrylamide gel electrophoresis. The DNA binding specificities of these proteins were similar and corresponded to that of the SEF1 binding activity in nuclear extracts. Each of these isolated SEF1 proteins also bound to the essential delta-E3 element of the human T cell receptor delta enhancer. Antibodies against one SEF1 protein only reacted with the protein used for immunization, which indicates a limited homology between at least some SEF1 proteins. We also present data suggesting that SEF1 proteins exist in multiple forms with differences in their DNA binding specificity, and that high affinity DNA binding of the SEF1 proteins requires protein phosphorylation.

Purification and Characterization of Cellobiose Dehydrogenase, a Novel Extracellular Hemoflavoenzyme from the White-Rot Fungus Phanerochaete chrysosporium

Cellobiose dehydrogenase (CDH), an extracellular hemoflavoenzyme produced by the cellulose-degrading cultures of Phanerochaete chrysosporium, oxidizes cellobiose to cellobionolactone. CDH has been purified to homogeneity by a five-step purification procedure. The homogeneous CDH is monomeric and has a relative molecular mass of 90,000. It is also a glycoprotein with a neutral carbohydrate content of 9.4%. Purified CDH contains one heme b and one flavin adenine dinucleotide per monomer. Homogeneous CDH has a specific activity of 10.3 μmol min−1 mg−1 for cytochrome c reduction, in the presence of cellobiose. Cellotriose, cellotetraose, cellopentaose, and lactose also serve as substrates for CDH, in addition to cellobiose. Cytochrome c, dichlorophenolindophenol, Mn3+, and benzoquinones can function as electron acceptors in these oxidations. Kinetic studies suggest that cellobiose is the preferred substrate and cytochrome c is the preferred electron acceptor. In the absence of these electron acceptors, oxygen serves as a poor electron acceptor and is reduced to H2O2. CDH is very stable in the pH range 3-10 and up to 50°C. At lower pH or at higher temperature, CDH is inactivated due to the release of flavin from the active site. The native ferric form of the enzyme has absorption maxima at 420, 529, and 570 nm. With the addition of cellobiose, these absorptions shift to 428, 534, and 564 nm. The ferric enzyme does not bind azide or cyanide, implying that the heme iron is probably hexacoordinate.