Purification of bovine glia maturation factor and characterization with monoclonal antibody

Glia maturation factor (GMF) is purified 100 000-fold to apparent homogeneity from bovine brains by a procedure consisting of ammonium sulfate precipitation, column chromatography with diethylaminoethyl-Sephacel, Sephadex G-75, and hydroxylapatite, and a final step using C4 reverse-phase high-performance liquid chromatography. The product shows a single protein band in sodium dodecyl sulfate-polyacrylamide gel. It has a molecular weight of 14 000 and an isoelectric point of pH 5.2. Purified GMF stimulates cultured astroblasts to proliferate and to grow out cell processes with half-maximal activity at 8 ng/mL. A monoclonal antibody raised against partially purified GMF adsorbs the activity of pure GMF and immunologically binds the putative GMF protein band.     

Purification and properties of cytochrome c-556 from Agrobacterium tumefaciens B2a.

Cytochrome c-556 from Agrobacterium mefaciens B2a was isolated in a pure, homoneous state. The best purification procedure volved ammonium sulphate fractionation, delting on Sephadex G-25, column chromatographic fractionation on DEAE- and CM-cellulose, and gel filtration on Sephadex G-75 superfine. Substitution of the CM-cellulose step by isoelectric focusing was successful. The purity of the final preparation is warranted by the purity index value, the electrophoretic patterns in the absence and presence of sodium dodecylsulphate, the sedimentation profile and the N-terminal amino acid analysis (alanine). The absorption spectrum of reduced cytochrome c-556 has maxima at 318, 419, 526 and 555.5 nm. The molar extinction coefficient for the alpha-band is 20 200M-1cm-1. The isoelectric point, determined both by preparative and analytical isoelectric focusing, is 5.55 +/- 0.10. The molecular weight of cytochrome c-556 was determined by gel filtration as 12000 and by dodecylsulphate gel electrophoresis as 11 500.     

A simplified and efficient procedure for the purification of apolipoprotein AI from human serum high-density lipoprotein-3 by preparative isoelectric focussing on polyacrylamide gel beads

An improved method is described for the purification of milligram amounts of apolipoprotein AI from serum apo-HDL₃ by isoelectric focussing on polyacrylamide gel beads. The procedure involves a single focussing over a narrow (1.3 unit) pH gradient, and permits isolation of apo-AI of exceptional purity and in high yield (75% recovery of HDL₃ protein, ca. 50% corresponding to pure apo-AI). The electrophoretic mobility, pI values, molecular weight, antigenicity and amino acid composition of such apo-AI were indistinguishable from those reported in the literature. A rabbit antiserum to apo-AI isolated by focusing exhibited similar immunological reactivity to one prepared from an antigen isolated by gel filtration chromatography; moreover, apo-AI purified by the respective procedures reacted identically with both antisera. We conclude that isoelectric focussing on a support of polyacrylamide gel beads (as Bio-Gel P60) presents certain advantages for the isolation of highly purified apo-AI over both conventional chromatographic procedures and isoelectric focussing on a Sephadex support.     

A simple method for the purification of murine corticosteroid binding globulin: characterization of a monomer

A fast and reproducible two-step method with high resolution was developed for purification of murine corticosteroid-binding globulin (CBG). The first step was liquid chromatography on a Sephacryl-S-200 column, and the CBG-containing residual was subsequently chromatographed by fast protein liquid chromatography (FPLC). This enabled us to quickly obtain a highly purified protein and the apparently isolated CBG was tested for its purity by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) and sedimentation equilibrium centrifugation. The CBG concentration in pregnant mouse serum was estimated to 0.78 g/l (1.5% of the total protein). The monomeric organization of the protein was demonstrated by mercaptoethanol treatment. No NH2-terminal amino acid could be detected, probably owing to a blocked amino acid. The mol. wt (Mr) of murine CBG was determined to be 52,000 and the sedimentation constant S20 degrees, w to 3.9 S by analytical ultracentrifugation. The protein showed 5 bands when subjected to isoelectric focusing: 3 bands with apparent isoelectric points (pI) between pH 3.15-3.25 and two between pH 3.40-3.50.     

A single-step purification of assembled insulin on a resource RPC column

We have designed an efficient single step purification process using Resource RPC column in conjunction with triethyl amine/acetonitrile solvent system at similar pH at which the in vitro assembly of insulin is carried out from its two chains. The conditions have been optimized to separate native insulin from its single chain precursors as well as from other by-products. This method obviates the need of acidification, centrifugation, gel filtration, and concentration steps before HPLC purification of insulin from assembly mixture. The system offers wide pH stability, rapid regenerability, excellent pressure/flow characteristics, and high loading capacity. The authenticity and purity of human insulin purified by this procedure was evaluated by analytical reverse phase HPLC, peptide mapping, and isoelectric focusing.     

Fractionation of horseradish peroxidase by preparative isoelectric focusing, gel chromatography and ion-exchange chromatography.

Horseradish peroxidase has been fractionated by preparative isoelectric focusing in a density gradient and in a layer of granulated gel using pH-3-10 and narrow-pH-range carrier ampholytes at different total enzyme loads. The resolution of peroxidase isoenzymes in preparative-layer isoelectric focusing was comparable to that obtained by analytical thin-layer isoelectric focusing. Isoelectrically homogeneous isoenzymes could be isolated with good recovery in a single fractionation step. Despite the excellent separation of the individual isoenzymes by isoelectric focusing in gel layers, an effective purification, indicated by the absorbance ratio A403mn/A278nm, could not be achieved by focusing applied as a single step. By different fractionation sequences combining gel chromatography, ion-exchange chromatography, and isoelectric focusing, individual isoenzymes with a high purity and homogeneous with respect to their size and charge properties have been isolated.     

明胶亲和层析介质分离纯化猪血浆纤维结合蛋白

目的：研究猪血浆中纤维结合蛋白的分离纯化方法,得到高纯度、高活性的目标蛋白。方法：以明胶亲和层析介质对猪血浆纤维结合蛋白进行分离纯化,考察了不同洗脱方式对纯化结果的影响,对产品进行了纯度、生物活性的测定。结果：通过对纯化工艺条件的摸索,采用含不同浓度尿素缓冲液进行分步洗脱,可以得到纯度为95％以上的产品,回收率达到50．96％;经BHK细胞培养实验证明其具有与人血浆纤维结合蛋白同样的生物活性。结论：采用了较温和的洗脱方式以明胶亲和层析介质对猪血浆纤维结合蛋白进行分离纯化,提高了动物资源的可利用性。     

Overexpression and purification of the galactose operon enzymes from Escherichia coli

A convenient new procedure for the purification of galactokinase, galactose-1-phosphate uridyltransferase, and UDP-galactose 4-epimerase overexpressed in Escherichia coli is presented. The procedure is shorter than any other described in the literature and facilitates the purification of the three recombinant enzymes in considerable amounts and at high purity and specific activity. The purified gal operon enzymes were biochemically characterized by gel-filtration column chromatography and isoelectric focusing, and the Km values for their substrates were determined.     

A novel scalable,robust downstream process for oncolytic rat parvovirus: isoelectric point-based elimination of empty particles

The rodent protoparvovirus H-1PV, with its oncolytic and oncosuppressive properties, is a promising anticancer agent currently under testing in clinical trials. This explains the current demand for a scalable, good manufacturing practice-compatible virus purification process yielding high-grade pure infectious particles and overcoming the limitations of the current system based on density gradient centrifugation. We describe here a scalable process offering high purity and recovery. Taking advantage of the isoelectric point difference between full and empty particles, it eliminates most empty particles. Full particles have a significantly higher cationic charge than empty ones, with an isoelectric point of 5.8–6.2 versus 6.3 (as determined by isoelectric focusing and chromatofocusing). Thanks to this difference, infectious full particles can be separated from empty particles and most protein impurities by Convective interaction media^® diethylaminoethyl (DEAE) anion exchange chromatography: applying unpurified H-1PV to the column in 0.15 M NaCl leaves, the former on the column and the latter in the flow through. The full particles are then recovered by elution with 0.25 M NaCl. The whole large-scale purification process involves filtration, single-step DEAE anion exchange chromatography, buffer exchange by cross-flow filtration, and final formulation in Visipaque/Ringer solution. It results in 98% contaminating protein removal and 96% empty particle elimination. The final infectious particle concentration reaches 3.5E10 plaque forming units (PFU)/ml, with a specific activity of 6.8E11 PFU/mg protein. Overall recovery is over 40%. The newly established method is suitable for use in commercial production.

     

A simple procedure for obtaining large quantities of tubulin subunits from newborn mouse brain

A simple, efficient two-step procedure of DEAE--Sephadex A--50 column chromatography followed by hydroxyapatite column chromatography for purification of alpha- and beta-tubulin subunits from newborn mouse brain is described. Stored frozen mouse brains can be used as a convenient starting material. Differential elution of alpha and beta subunits from hydroxyapatite was achieved using a linear gradient of 0.2--0.3 M phosphate buffer containing 2 M urea, 0.1% sodium dodecyl sulphate and 1 mM dithiothreitol. 20 mg of alpha-tubulin (purity: greater than 85%) and 16 mg of beta-tubulin (purity greater than 95%) completely separated from each other can be obtained in one experiment.     

Novel method for purification of staphylococcal enterotoxin A.

A novel single-step procedure for the purification of staphylococcal enterotoxin A (SEA), namely, dye ligand affinity chromatography with the triazine dye Red A, was developed. SEA purified by this method produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield from 5 liters of culture supernatant was 0.113 g, corresponding to an overall yield of 55%. In some instances, purification of SEA from culture supernatants by dye ligand affinity chromatography produced two enterotoxin peaks that could be eluted from the column with 300 and 500 mM phosphate buffer (pH 6.8). Enterotoxin from these peaks produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but multiple bands were observed on isoelectric focusing gels. This method of purification represents a significant improvement in time, yields, and purity of enterotoxin over previously published purification methods.     

Novel method for purification of staphylococcal enterotoxin A

A novel single-step procedure for the purification of staphylococcal enterotoxin A (SEA), namely, dye ligand affinity chromatography with the triazine dye Red A, was developed. SEA purified by this method produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield from 5 liters of culture supernatant was 0.113 g, corresponding to an overall yield of 55%. In some instances, purification of SEA from culture supernatants by dye ligand affinity chromatography produced two enterotoxin peaks that could be eluted from the column with 300 and 500 mM phosphate buffer (pH 6.8). Enterotoxin from these peaks produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but multiple bands were observed on isoelectric focusing gels. This method of purification represents a significant improvement in time, yields, and purity of enterotoxin over previously published purification methods.     

Glia Maturation Factor Expression in Hippocampus of Human Alzheimer’s Disease

Alzheimer’s disease (AD) is characterized by the presence of neuropathological lesions containing amyloid plaques (APs) and neurofibrillary tangles (NFTs) associated with neuroinflammation and neuronal degeneration. Hippocampus is one of the earliest and severely damaged areas in AD brain. Glia maturation factor (GMF), a known proinflammatory molecule is up-regulated in AD. Here, we have investigated the expression and distribution of GMF in relation to the distribution of APs and NFTs in the hippocampus of AD brains. Our immunohistochemical results showed GMF is expressed specifically in the vicinity of high density of APs and NFTs in the hippocampus of AD patients. Moreover, reactive astrocytes and activated microglia surrounds the APs and NFTs. We further demonstrate that GMF immunoreactive glial cells were increased at the sites of Tau containing NFTs and APs of hippocampus in AD brains. In conclusion, up-regulated expression of GMF in the hippocampus, and the co-localization of GMF and thioflavin-S stained NFTs and APs suggest that GMF may play important role in the pathogenesis of AD.     

Single-step method for purification of Shiga toxin-1 B subunit using receptor-mediated affinity chromatography by globotriaosylceramide-conjugated octyl sepharose CL-4B.

A new single-step purification method for Shiga toxin (Stx) was developed using receptor-mediated affinity chromatography, in which Gb3Cer (globotriaosylceramide) was conjugated to octyl Sepharose CL-4B as a carrier. This method achieves high yield and high purity in a small column on which Gb3Cer has been immobilized at high density. Using this affinity column, the Stx1 B subunit was purified with homogeneity by a one-step procedure from a crude extract of recombinant Stx1 B subunit-producing Escherichia coli. The purified Stx1 B subunit conserved a natural pentamer structure confirmed by gel filtration and sedimentation equilibrium analysis. Furthermore, the purified Stx1 B subunit was able to bind specifically to Gb3Cer expressed on Burkitt's lymphoma cells. This versatile purification method can be used to isolate various types of natural as well as recombinant Stx, facilitating fundamental studies of human diseases caused by this toxin.     

Isolation of highly purified cyclodextrin glucanotransferase from Bacillus sp. 1070

Two chromatographic processes for purification of cyclodextringlucanotransferase (CGTase) from Bacillus sp. 1070 was carried out. The enzyme has been purified into 9.5 times on Butyl-Toyopearl and followed immobilized metal ion chromatography on Cu(II)-Iminodiacetic (IDA)-agarose. By the application of second purification scheme (chromatography on Butyl-Toyopearl and DEAE-Sephacel) the specific activity of CGTase has folded into 13.5 times. The purity of enzyme was shown to be approximately 90% by SDS-electrophoreses data. It was shown that isolated enzyme has two isoelectric points estimated as 5.1 and 5.3.     

Purification and characterization of human kidney renin

By means of a new rapid and small scale purification method, human kidney renin has been purified from a single kidney in a homogeneous state, as judged on SDS-PAGE. The kidney which showed unusually high renin activity was from a patient with cardiomyopathy. 8,000-fold purification was attained by means of only pepstatin-aminohexyl-Sepharose chromatography and FPLC on a Mono Q column, and the yield was 34%. The specific activity was 5.63 mg angiotensin I per mg protein per h at 37 degrees C and pH 6.5 with porcine angiotensinogen as the substrate. The molecular weight was estimated to be 37,000 by SDS-PAGE and 38,000 by HPLC on a TSK G-3000 SW column. The preparation showed three bands on isoelectric focusing. The molecular weight and the profile on isoelectric focusing of the purified renin agreed with those found for the extracts of both the patient's kidney and a kidney with the usual low renin activity.     

Large-scale recovery of C-phycocyanin from Spirulina platensis using expanded bed adsorption chromatography

C-phycocyanin was purified on a large scale by a combination of expanded bed adsorption, anion-exchange chromatography and hydroxyapatite chromatography from inferior Spirulina platensis that cannot be used for human consumption. First, phycobiliproteins were extracted by a simple, scaleable method and then were recovered by Phenyl-Sepharose chromatography in an expanded bed column. The purity (the A(620)/A(280) ratio) of C-phycocyanin isolated with STREAMLINE column was up to 2.87, and the yield was as high as 31 mg/g of dried S. platensis. After the first step, we used conventional anion-exchange chromatography for the purification steps, with a yield of 7.7 mg/g of dried S. platensis at a purity greater than 3.2 and with an A(620)/A(650) index higher than 5.0. The fractions from anion-exchange chromatography with a level of purity that did not conform to the above standard were subjected to hydroxyapatite chromatography, with a C-PC yield of 4.45 mg/g of dried S. platensis with a purity greater than 3.2. The protein from both purification methods showed one absolute absorption peak at 620 nm and a fluorescence maximum at 650 nm, which is consistent with the typical spectrum of C-phycocyanin. SDS-PAGE gave two bands corresponding to 21 and 18 kDa. In-gel digestion and LC-ESI-MS showed that the protein is C-phycocyanin.     

Isoelectric precipitation and gel chromatography for purification of monoclonal IgM

A preparative scale purification procedure of monoclonal IgM from hybridoma culture supernatant with high protein content is described. The procedure consists of three steps starting with ultrafiltration followed by isoelectric precipitation and gel chromatography. Cells and debris from culture supernatant were removed by microfiltration. The clarified supernatant was concentrated 400-fold in a hollow fibre ultrafiltration apparatus (cut off 100 000 daltons). The concentrate was titrated with dilute histidine/HCl buffer close to the isoelectric point of the IgM. Precipitated proteins were harvested by centrifugation, washed and redissolved. The protein fraction containing the IgM was further purified by gel chromatography on a Sephacryl S300 column. This procedure leads to product recovery of 40% and purity of 99% related to total protein.     

Purification and analysis of the major components of chum salmon protamine contained in insulin formulations using high-performance liquid chromatography

A simple high-performance liquid chromatographic method has been developed for the rapid purification and analysis of protamine components contained in insulin formulations. Only a single step is needed to separate peptides whose compositions, sizes, and unusual isoelectric points (pI 13.8) are nearly identical. The method involves their isocratic separation on a reversed-phase column using a pH 2 phosphate buffer and a low acetonitrile content as an eluant. The purified chum salmon components were analyzed by amino acid analysis, solid-phase amino acid sequencing, carboxypeptidase B digests, insulin complexation analysis, and a mass spectrophotometric procedure which gives an accurate mass of the intact peptides. This HPLC purification technique may also be applicable to protamines and other highly basic peptides isolated from other sources.