Purification of phosphatidylinositol kinase from bovine brain myelin.

A membrane-bound phosphatidylinositol (PI) kinase (EC 2.7.1.67) was purified by affinity chromatography from bovine brain myelin. This enzyme activity was solubilized with non-ionic detergent and chromatographed on an anion-exchange column. Further purification was achieved by affinity chromatography on PI covalently coupled to epoxy-activated Sepharose, which was eluted with a combination of PI and detergent. The final step in the purification was by gel filtration on an Ultrogel AcA44 column. This procedure afforded greater than 5500-fold purification of the enzyme from whole brain myelin. The resulting activity exhibited a major silver-stained band on SDS/polyacrylamide-gel electrophoresis with an apparent Mr 45,000. The identity of this band as PI kinase was corroborated by demonstration of enzyme activity in the gel region corresponding to that of the stained protein. The purified enzyme exhibited a non-linear dependence on PI as substrate, with two apparent kinetic components. The lower-affinity component exhibited a Km similar to that observed for the phosphorylation of phosphatidylinositol 4-phosphate by the enzyme.     

Purification and chemical characterization of human hexosaminidases A and B.

N-Acetyl-beta-hexosaminidases A and B were purified to homogeneity from human placenta. In the initial step of purification, the enzymes were adsorbed on concanavalin A-Sepharose 4B and eluted from the column with alpha-methyl D-mannosides. Subsequent purification steps included DEAE-cellulose column chromatography, QAE-Sephadex [diethyl-(2-hydroxypropyl)aminoethyl-Sephadex] column chromatography, Sephadex G-200 gel filtration and preparative disc polyacrylamide-gel electrophoresis, followed by another QAE-Sephadex chromatography for the hexosaminidase A preparation, and DEAE-cellulose column chromatography, calcium phosphate gel chromatography, Sephadex G-200 gel filtration, QAE-Sephadex chromatography and CM-cellulose chromatography for the hexosaminidase B preparation. The purified preparations, particularly hexosaminidase A, had significantly higher specific enzyme activities than previously reported. The preparations moved on polyacrylamide-gel electrophoresis as single protein bands, which also stained for enzyme activity. Sedimentation-equilibrium centrifugation indicated homogenous dispersion of the enzymes, and the molecular weight was estimated as about 110000 for both enzymes. Complete amino acid and carbohydrate compositions of the two isoenzymes were determined, and, in contrast with previous suggestions, no sialic acid was found in the enzymes.     

Human alpha-fetoprotein /AFP/ I. Isolation of homogenous AFP from cord blood serum

Summary The AFP from human cord blood was isolated by means of affinity chromatography with the use of antibodies as ligands and by gel filtration. The preliminary purification was achieved by affinity chromatography on CNBr-Sepharose 4B coupled with anti AFP-antibody. Further purification was obtained by the use of immunoadsorbent with anti-human serum protein antibodies. Final purification was achieved by gel filtration on Sephadex G-200. Homogeneity of the purified AFP was demonstrated by means of gel filtration, polyacrylamide gel electrophoresis, isoelectric focusing and immunoelectrophoresis.Supported by Polish National Cancer Programm within the project PR 6 0227/02/.     

Purification and heterogeneity of human kininogen. Use of DEAE-chromatography, molecular sieving and antibody specific immunosorbents.

Various methods of preparing human kininogen were investigated with an aim to limit the immunoreactive contaminant proteins to permit purification by immunosorption. A five-step procedure is described giving 7.5% yield of highly purified kininogen (pharmacological purity 14--20) from pooled human plasma, and containing approximately 30% alpha-2HS-glycoprotein and 2.8% albumin. Alpha-2HS could not be removed by polyacrylamide gel electrophoresis or isoelectric focusing in column. Analysis of heterogeneity of kininogen after chromatography on DEAE-Sephadex using various linear gradients and gel filtration on Sephadex G-100 suggested that a minor component may be an aggregate, not included in the yield. It remains uncertain whether this component derives from an occasionally observed high molecular form of active kininogen in the primary purification steps in the 7-12 S sieve fractions from Sephadex G-200, and excluded from further purification by pooling. Purification with immunosorbents was investigated using batch operations with antibody specific polymers prepared with antisera insolubilized with ethylchloroformate. It was found that the adsorption-desorption procedure was favourable for immunization purposes in producing highly specific immunologically pure kininogen. The kininogen obtained by this method or by the removal of contaminant alpha-2HS and albumin with the corresponding antibody specific polymers gave similar heterogenous patterns by polyacrylamide gel electrophoresis, indicating a main band of kininogen and several faintly stained bands which responded only to anti-kininogen. With 200 mug of the kininogen protein purified by immunosorption using monospecific antiserum the kininogen precipitation titre was 1:8 after 6--8 weeks in rabbits. With a polymer prepared with 4 ml anti-kininogen serum (1:8) and incubated with 800 mug highly purified kininogen approximately half the protein was desorbed with 2 M and 3 M sodium iodide in the first adsorption-desorption procedure.     

Enzymes Produced by a Pseudomonas Species Which Inactivate Inhibitors of Certain Viral Hemagglutinins. I. Identification and Purification of a Proteinase and Phospholipase C

Filtrates from cultures of a psychrophilic Pseudomonas species, which inactivate serum inhibitors of certain viral hemagglutinins, were shown to contain both lecithinase (phospholipase C) and a proteolytic enzyme with elastase activity. The bacterium was cultivated under conditions favoring production of the respective enzymes, and the enzymes were purified by ammonium sulfate precipitation followed by column chromatography or by gel filtration. The elastase was obtained in crystalline form and was recrystallized. It has properties similar to those of a number of other bacterial elastases but is more heat-labile than most. Although a high degree of purification was achieved for the lecithinase, as evidenced by an increase in specific activity, it was not obtained in crystalline form. Partially purified preparations of the lecithinase had extremely high activity compared to that of commercial preparations of phospholipase C from Clostridium welchii.     

鲍曼不动杆菌烈性噬菌体的分离与纯化

利用柱层析方法,纯化鲍曼不动杆菌（Acinetobacter baumannii）烈性噬菌体AB1。首先采用聚乙二醇6000沉淀方法,初步分离裂解液中的噬菌体,噬菌体纯度由6.1×1010 pfu/mg提高到37×1010 pfu/mg,噬菌体回收率为58.8%,蛋白质去除率为90.6%;噬菌体粗提样品经Sepharose 4B凝胶过滤层析柱进一步纯化,纯度提高到73×1010 pfu/mg,噬菌体回收率为95.7%,蛋白质去除率为48.1%;收集的噬菌体样品最后经DEAE-52阴离子交换层析柱处理,噬菌体纯度为40×1010 pfu/mg,回收率为50.8%,蛋白去除率15.6%。内毒素分析结果显示,Sepharose 4B凝胶过滤层析纯化的噬菌体样品中,内毒素含量为443.8 EU/mg,而DEAE-52阴离子交换层析纯化的噬菌体样品中,内毒素含量为544.4 EU/mg。实验结果显示,PEG沉淀方法与Sepharose 4B凝胶过滤方法能够有效地提高噬菌体纯度,而DEAE-52阴离子交换层析则不能提高噬菌体的纯度,也无法有效地去除样品中的内毒素。     

Rapid, simplified method for production and purification of tetanus toxin

A rapid, simplified method for production and purification of tetanus toxin from bacterial extracts was described. The extracts were prepared by stirring young cells (ca. 45-h culture) of Clostridium tetani in 1 M NaCl-0.1 M sodium citrate, pH 7.5, overnight at 0 to 4 degrees C. The toxin was purified by a combination of (i) ammonium sulfate fractionation (0 to 40% saturation), (ii) ultracentrifugation for removal of particulate materials, and (iii) gel filtration by high-pressure liquid chromatography on a TSK G3000 SW-type column. This method required 6 days as follows: (i) overnight incubation of the seed culture, (ii) 2 days for growing the bacteria for toxin production, (iii) overnight extraction of the toxin from the bacteria, (iv) overnight precipitation of the toxin with ammonium sulfate, (v) 2 h for ultracentrifugation of the ammonium sulfate concentrate of the bacterial extract, and (vi) 1 h for high-pressure liquid chromatography. The minimum lethal dose of the purified toxin preparations for mice was 1.4 X 10(7) to 1.5 X 10(7) per mg of protein and they showed 360 to 390 Lf (flocculating activity) per mg protein and a 280/260 nm absorbance ratio of 2.0 to 2.1. The final recovery of the toxin from bacterial extracts was 90 to 93%. The purified preparations gave a single band of toxin protein with a molecular weight of 150,000 +/- 5,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On crossed immunoelectrophoresis, the purified toxin preparations gave a single precipitation arc against anti-crude toxin serum.     

Rapid, simplified method for production and purification of tetanus toxin.

A rapid, simplified method for production and purification of tetanus toxin from bacterial extracts was described. The extracts were prepared by stirring young cells (ca. 45-h culture) of Clostridium tetani in 1 M NaCl-0.1 M sodium citrate, pH 7.5, overnight at 0 to 4 degrees C. The toxin was purified by a combination of (i) ammonium sulfate fractionation (0 to 40% saturation), (ii) ultracentrifugation for removal of particulate materials, and (iii) gel filtration by high-pressure liquid chromatography on a TSK G3000 SW-type column. This method required 6 days as follows: (i) overnight incubation of the seed culture, (ii) 2 days for growing the bacteria for toxin production, (iii) overnight extraction of the toxin from the bacteria, (iv) overnight precipitation of the toxin with ammonium sulfate, (v) 2 h for ultracentrifugation of the ammonium sulfate concentrate of the bacterial extract, and (vi) 1 h for high-pressure liquid chromatography. The minimum lethal dose of the purified toxin preparations for mice was 1.4 X 10(7) to 1.5 X 10(7) per mg of protein and they showed 360 to 390 Lf (flocculating activity) per mg protein and a 280/260 nm absorbance ratio of 2.0 to 2.1. The final recovery of the toxin from bacterial extracts was 90 to 93%. The purified preparations gave a single band of toxin protein with a molecular weight of 150,000 +/- 5,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On crossed immunoelectrophoresis, the purified toxin preparations gave a single precipitation arc against anti-crude toxin serum.     

Purification, potency and immunogenicity analysis of Vero cell culture-derived rabies vaccine: a comparative study of single-step column chromatography and zonal centrifuge purification

Continuous Vero cell lines are more suitable for large-scale production of rabies vaccine. The purification of Vero cell-derived rabies vaccine is critical because of the residual cellular DNA and serum proteins. The perfection of techniques using column chromatography with different matrix material, gel filtration and zonal centrifugation is of paramount importance for the optimal purification of rabies vaccine, leaving minimal residual cellular DNA, below the permissible level of 100 pg per dose and serum protein content of 1 ppm. In this study the potency, immunogenicity and safety of Vero cell-derived rabies vaccines were compared following purification by densely or loosely packed DEAE-sepharose CL-6B columns with different bed heights or by zonal centrifugation. The optimal virus recovery and maximum removal of substrate DNA and serum proteins were achieved only when the sepharose CL-6B column bed height was maintained at a thickness of 2-2.5 cm. The rabies virus material was purified by layering over the matrix without applying pressure. DEAE-sepharose CL-6B column purification using a simplified, cost effective technique as described in this study enhances the antigen yield by 50% in comparison with zonal purification.     

Purification of VDAC (Voltage-dependent anion-selective channel) from rat liver mitochondria

The outer membrane of rat liver mitochondria contains a channel-forming protein known as VDAC (voltage-dependent anion-selective channel). This protein has been functionally purified by a combination of ion exchange chromatography, gel filtration and affinity chromatography on a Concanavalin A-containing column. An estimated 300-fold purification was achieved over the specific activity in mitochondrial membranes. When the purified protein is run on an SDS polyacrylamide gel, essentially only one band is present at a position consistent with a molecular weight of 32,000. The resulting protein is functional and behaves normally based on channel size, selectivity and voltage dependence.     

Purification, stabilization, and characterization of rat hepatic triglyceride lipase

Hepatic triglyceride lipase (H-TGL) was purified to near homogeneity from heparin-containing rat liver perfusates with the following column chromatography steps: heparin-Sepharose affinity chromatography, anion-exchange chromatography on DEAE-Sephacel, and gel filtration on Ultrogel AcA 34. A final specific activity of 45,000 μmol fatty acid/mg/h was obtained with an overall 31% recovery of catalytic activity. The heparin-Sepharose step resulted in a 20-fold purification, while the DEAE and gel filtration steps led to further purification with complete recovery of activity. An extensive survey of various detergents as potential stabilizers of H-TGL activity led to the selection of Triton N-101 for use in the column buffers of the DEAE and gel filtration steps. Relative to initial H-TGL activity upon dilution in buffer without detergent, recoveries between 90 and 100% were consistently obtained with Triton N-101-containing buffers following a 24-h incubation at 20°C. In contrast after a 24-h incubation at 20°C those control samples lacking detergent were at least 95% inactivated. The highly purified H-TGL exhibited a single major band by sodium dodecyl sulfate-electrophoresis. The use of DEAE chromatography and stabilization of H-TGL with Triton N-101 are the improvements in purification that resulted in an 8-fold enhancement in specific activity relative to the highest previous report of purification from rat liver perfusates.     

Properties of Purified Staphylococcal β-Hemolysin

Purification of beta-hemolysin was achieved by ammonium sulfate precipitation, Sephadex G-100 gel filtration, carboxymethyl cellulose column chromatography, and density gradient electrophoresis. Active fractions eluted from carboxymethyl cellulose contained at least one nonhemolytic protein, and omission of this step was not detrimental to the purification process. Density gradient electrophoresis yielded approximately 1.6 mg of highly active purified beta-hemolysin per liter of culture supernatant liquid. Purified beta-hemolysin gave a single line on gel double diffusion and immunoelectrophoresis. A single symmetrical peak formed in the analytical ultracentrifuge, and the sedimentation coefficient was calculated to be 1.7S. The purified beta-hemolysin was stable at 4 C and could be lyophilized. Magnesium cations were required for full expression of beta-hemolytic activity. beta-Hemolysin was lethal for rabbits when injected intravenously in amounts between 40 and 160 mug. Crude beta-hemolysin was more stable than purified beta-hemolysin when heated at 60 C for 30 min. Purified beta-hemolysin lost almost all of its activity on subsequent heating at 100 C for 10 min.     

C1q: isolation from human serum in high yield by affinity chromatography and development of a highly sensitive hemolytic assay.

C1q, a subcomponent of the first component of complement, has been isolated from human serum in fully hemolytically active form by affinity column chromatography and gel filtration with Bio-Gel A-5M. The affinity column was prepared by covalent coupling of purified human IgG to CNBr-activated Sepharose 4B. Final yields of C1q ranged from 25 to 40% with 650- 890-fold purification based on recovery of hemolytic activity. The preparations were free of contaminating serum proteins as judged by SDS-polyacrylamide gel electrophoretic and immunochemical criteria. The final C1q preparations were also devoid of any demonstrable C1q-inhibitor activity. A C1q-depleted reagent (C1qD) was obtained from the nonabsorbed protein containing fractions of the human IgG-Sepharose 4B affinity column and utilized in conjunction with sensitized sheep erythrocytes (EA) for the detection and quantitation of C1q hemolytic activity. Employing optimal quantities of C1qD in the hemolytic assay mixture, the highly purified C1q preparations contained 0.5 to 1 x 10(13) effective molecules/mg and 0.5 to 1 x 10(12) effective C1q molecules/ml of human serum. This assay would therefore reproducibly detect less than 1 ng of C1q hemolytic activity.     

Characterization of a Phenol Oxidase from Cryptococcus neoformans var. neoformans

In Cryptococcus neoformans, enzymic oxidation of various catechols leads to melanin, a proposed virulence factor. A phenol oxidase enzyme of Cryptococcus neoformans var. neoformans produced at 25 C has been purified from an ultracentrifugal supernatant of an extract of broken cells. Hydrophobic interaction chromatography followed by anion-exchange column chromatography allowed purification of the phenol oxidase. The molecular weight of the enzyme estimated by gel filtration was about 80,000 and a dimeric species (Mw = 160,000) was suggested. The isoelectric point of the protein was approximately 4.1. An NH₂-terminal 31 amino acid sequence was determined using phenol oxidase electroblotted onto a PVDF membrane after nondenaturing gel electrophoresis. Upon searching the Peptide Institute (Osaka) data base, no proteins with high degrees of homology were found.     

An automatic multidimensional chromatography system for purification of human uterine progesterone receptor and induction of polyclonal antibodies

This paper reports on the synthesis of Org2058-bonded microparticulate silicas and their use in affinity chromatography as the first step for the purification of human progesterone receptor. The development of microprocessor-controlled instruments allows all the various steps to be performed automatically. The various steps used for the purification of human progesterone receptor were carried out with the FPLC system: affinity chromatography, desalting of eluate on Sephadex G-25, anion-exchange chromatography using a Mono Q column. With this procedure the receptor was purified approx. 10,000-fold within 24 h. The yield of receptor was generally 85-95%. Investigations with induced anti-progesterone receptor antibodies obtained after the fourth immunization show their immunoreactive behaviour towards progesterone receptor in crude cytosol, which was proved by sucrose density gradient centrifugation and by gel filtration on the FPLC system using a Sepharose 12 column. This implies that progesterone receptor was efficiently purified by our purification procedure.     

Purification and properties of chicken egg-white cobalamin-binding protein

A cobalamin-binding protein has been purified from chicken egg-white by using a combination of conventional and high performance ion-exchange chromatography. Following initial purification by DEAE-cellulose, ammonium sulphate precipitation, Sephacryl S-200 CM-cellulose and affinity chromatography, appropriate fractions were further purified using the Pharmacia fast protein liquid chromatography (FPLC) system. Using this method of purification, egg-white CBP has been purified more rapidly and with greater recovery than with conventional column chromatography. The homogeneity of this protein was verified by SDS-PAGE. The Mr was 37,000 by SDS-PAGE and 39,000 by gel filtration, which indicated that it was a glycoprotein. The stokes radius was 4.1 nm and pI was 4.3. The protein bound 57COB12 with a molar ratio of 1/1 and kd of 0.40 microM. The egg-white CBP was composed of 294 amino acid residues. Thiol groups and metal ions were not connected with the Cbl-binding activities.     

Expression and purification of hexahistidine-tagged human glutathione S-transferase P1-1 in Escherichia coli

The bacterial expression and purification of human pi class glutathione S-transferase (hGST P1-1) as a hexahistidine-tagged polypeptide was performed. The expression plasmid for hGST P1-1 was constructed by ligation of the cDNA which codes for the protein into the expression vector pET-15b. The expressed protein was purified by either glutathione or metal (Co(2+)) affinity column chromatography, which produced the pure and fully active enzyme in one step with a yield of more than 30 mg/liter culture. The activity of the purified protein was 130 units mg(-1) from the GSH affinity column and 112 units mg(-1) from the Co(2+) affinity column chromatography. The purity of the protein was assessed by electrospray ionization mass spectrometry and size-exclusion chromatography. It showed that the real molecular weight of the hexahistidine-tagged hGST P1-1 polypeptide chain agreed with the calculated value and that the purified protein eluted as an apparent homodimer on the gel filtration column. Our expression system allows the expression and purification of active hexahistidine-tagged hGST P1-1 in high yield with no need of removal of the hexahistidine tag and gives pure protein in one purification step allowing further study of this enzyme.     

Purification of a specific repressor of ferritin mRNA translation from rabbit liver

The synthesis of ferritin is regulated at the translation level in coordination with iron availability. Under conditions of low iron, translation of ferritin mRNA is repressed and the majority of ferritin mRNA is non-polysomal. Upon an increase in iron, translation of ferritin mRNA is derepressed resulting in as much as a 50-100-fold increase in the rate of ferritin synthesis. This regulation is mediated at least in part by a specific translational repressor which binds to a conserved sequence, the iron responsive element, located in the 5'-untranslated region of ferritin mRNA. In this communication we report the purification of such a repressor from rabbit liver. This repressor, which we call the "ferritin repressor protein," has an apparent molecular mass of 90 kDa when analyzed by gel filtration chromatography. It inhibits translation of ferritin mRNA in a highly specific fashion when added to a wheat germ lysate programmed with liver poly(A+) mRNA. In addition, it binds specifically to sequences contained within the first 92 nucleotides of ferritin mRNA, most likely the iron responsive element. Analysis of highly purified repressor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it is composed primarily of a single polypeptide of approximately 90 kDa. Elution of this 90-kDa polypeptide from a sodium dodecyl sulfate gel followed by renaturation and analysis for repressor activity shows that it both binds to the 5'-untranslated region of ferritin mRNA and represses its translation in vitro.     

Isolation of alpha-lactalbumin,beta-lactoglobulin,and bovine serum albumin from cow's milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity

The aim of this study was to introduce a simple, reproducible, and less expensive method for isolation of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin from cow's milk while retaining their antigenicity. Whey (lactoserum) was obtained by isolating casein from defatted milk using hydrochloric acid. Globulins were then precipitated from whey by half-saturated ammonium sulfate and beta-lactoglobulin was purified further using Sephadex G-50 gel filtration. The proteins in the supernatant were also fractionated using diethylaminoethyl cellulose chromatography in which beta-lactoglobulin was separated from alpha-lactalbumin and bovine serum albumin. The latter two proteins that co-eluted in anion-exchange chromatography were then gently isolated from each other by Sephadex G-50 gel filtration. Pure beta-lactoglobulin was also obtained by anion-exchange chromatography of the ammonium sulfate-precipitated globulins. Using enzyme-linked immunosorbent assay (ELISA), Western blotting, and ELISA inhibition assay, antigenicity of the purified proteins was evaluated. Our results showed high purity and well-preserved antigenicity of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin thus purified.     

Purification of glucoamylase by acarbose (BAY g-5421) affinity chromatography

Aspergillus niger and Rhizopus sp. glucoamylases were purified on an affinity chromatography column from commercially available, impure enzyme preparations. Up to 2 mg of glucoamylase protein was bound without leakage to a 1-ml affinity gel column (0.7 X 2.5 cm) possessing a covalently linked acarbose ligand (1 mg acarbose/g wet gel), and the bound enzyme was specifically released by irrigation of the column with a solution of maltose. A complete cycle of purification was accomplished in about 8 h. Glucoamylases were recovered, in more than 80% yield, free of alpha-amylase activity and possessing specific activities comparable to those of preparations obtained by time-consuming, multistep procedures involving several ion-exchange and hydrophobic column fractionations. Thus, acarbose affinity chromatography provides a general method for the rapid and efficient purification of the glucoamylases, and seems to be ideally suited for scale-up for the commercial purification of these enzymes.