CL-6B Column Research Articles

Establishment of a simplified dichotomic size-exclusion chromatography for isolating extracellular vesicles toward clinical applications.

Size‐exclusion chromatography (SEC) is a widely adopted method for the isolation of extracellular vesicles (EVs) from complex samples. SEC can efficiently remove high‐abundant proteins, while often requires multiple fractionation operation using diversified column settings. In this study, we aim to establish a simplified SEC method to acquire high quality EVs. In comparison of all three cross‐linked Sepharose resins with the sample types of FBS and human serum (HS), CL‐6B and CL‐4B showed superior performance in regular SEC to CL‐2B in terms of significantly narrower EV and protein peaks, higher resolutions and EV purity. By increasing their bed volumes to 20 ml, the resolutions of CL‐6B and CL‐4B columns could be significantly improved, while the CL‐6B column had the best performance with higher particle yields and tighter EV peaks. With the CL‐6B 20 ml column, we further established a simplified dichotomic SEC method that only requires two bulk elutions to acquire EVs in the Eluate 1 and proteins in the Eluate 2. We further justified that such CL‐6B columns were reusable for at least 10 consecutive times, and the dichotomic SEC was applicable to EV isolations from HS and FBS‐free supernatants of fluorescently labelled and unlabelled SW620 cells. The proteomics analysis implicated that although the two methods had dissimilar abilities in removing different co‐isolating contaminant proteins from EVs, the dichotomic SEC and ultracentrifugation could isolate EVs from human plasma with comparable purity. This dichotomic SEC has its intriguing potential to be used for EV preparation toward clinical testing and/or basic research.

Purification and activity determination of native and recombinant HBHA protein

Objective To purify native and recombinant heparin-binding hemagglutinin(HBHA)protein,and investigate the activity of HBHA polyclonal antibody against aggregation of Bacillus CalmetteGuerin(BCG)induced by HBHA.Methods After growing BCG to the stationary phase in the 7H9 liquid medium,the native HBHA protein(nHBHA)was obtained by CL-6B column chromatography.At the same time,the HBHA gene fragment was cloned and expressed by transforming Escherichia coli BL-21.Then the polyclonal antibody against rHBHA was prepared by immunizing rabbit.Different comcentration of the HBHA protein was added to the BCG liquid medium,and the aggregation of the BCG was observed.Then,add the HBHA protein that incubated with anti-HBHA antibodies to the BCG culture medium and observe the aggregation of BCG.Results The purity of native HBHA was 99% and the concentration was 1.016 mg/ml.The expressed product contained 36% of total somtic protein.After purified,the purity of the recombinant HBHA protein was 97.1% and the concentration was 10.98 mg/ml.Both the rHBHA and nHBHA could induce the aggregation of BCG.When then concentration of nHBHA is 0.2μg/ml,BCG could be induced to aggregate,while the rHBHA concentration is 2μg/ml could induce the aggregation.Both aggregations could be suppressed by the polyclonal antibody against rHBHA.Conclusions The native and recombinant HBHA are successfully obtained.It is proved that the rHBHA could induce the aggregation of BCG similar as nHBHA,and polyclonal antibody against rHBHA could also suppress the activity of nHBHA.It suggested that rHBHA could be further used in clinical diagnosis and vaccination. Key words: Mycobacterium tuberculosis; Lectins; Antibodies

Setaria cervi: in vitro released collagenases and their inhibition by Wuchereria bancrofti infected sera.

In vitro released products of adult Setaria cervi females, microfilariae and extracts showed considerable amounts of collagenase activity. On the basis of per mg protein released in vitro, the products of both microfilariae and adult females exhibited comparable activity but this was much higher than that of extract of microfilariae and adult females. Two collagenase enzymes with molecular masses of 50 kDa and 70 kDa were separated using DEAE-sepharose CL6B and Sephadex G-100 column chromatography. The 50 kDa and 70 kDa collagenase exhibited pH optima of 5.2 and 7.0, respectively. Considering specific activity, the 50 kDa enzyme was found to contribute about ten times more collagenase activity as compared to the 70 kDa enzyme. An inhibition study revealed obvious differences between them. Thiol group inhibitors such as N-ethylmaleimide and leupeptin inhibited the 50 kDa enzyme but this was strongly activated by dithiothreitol, a thiol group stabilizer. Alternatively, the 70 kDa enzyme showed a sensitivity to a metal chelator and a serine group inhibitor indicating its metalloserine protease nature. The antifilarial drug diethylcarbamazine did not demonstrate any inhibition under in vitro conditions. Both enzymes were significantly inhibited by antibody IgG separated from Wuchereria bancrofti infected human sera, showing a possible immunoprotective role.

Purification and kinetic properties of betaine-homocysteine methyltransferase from Aphanothece halophytica.

Betaine-homocysteine methyl transferase (BHMT) from Aphanothece halophytica was purified to homogeneity by hydroxyapatite, DEAE-Sepharose CL-6B and Sephadex G-200 column chromatography. A 24-fold purification and 11% overall yield were achieved with a specific activity of 595 nmol h(-1) mg(-1). The subunit molecular weight was determined to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the native enzyme was found to have a molecular weight of 350 kDa, suggesting an octameric structure of the enzyme. The enzyme shows optimum activity at 37 degrees C, pH 7.5. The apparent Km values for glycinebetaine and L-homocysteine were 4.3 mM and 1.3 mM, respectively. The enzyme was 70% inactivated by 5 mM dimethylglycine whereas the same concentration of sarcosine slightly inactivated the enzyme. Two analogs of glycinebetaine were also tested for enzyme inactivation and it was found that 5 mM choline inactivated 60% of the enzyme activity and 2.5 mM betaine aldehyde completely abolished the enzyme activity. NaCl at 200 mM or higher also completely inactivated the enzyme.

Different selenium-containing proteins in the extracellular and intracellular media of leucocytes cultivated in vitro.

The purpose of this communication is to elucidate if selenium plays a role in the function of granulocytes and lymphocytes. Thus, the incorporation of selenium in proteins from granulocytes and lymphocytes cultured with 1 microCi/mL radioactive Na2(75)SeO3 was studied. The protein peaks containing 75Se from two columns of Heparin Sepharose CL-6B and Sephacryl S-200 HR were separated further by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results showed that the incorporation of 75Se into granulocytes was about six times higher than that of lymphocytes during a 96-h cultivation, however, the GSH-Px activity in granulocytes did not change significantly. On the other hand, the GSH-Px activity of lymphocytes rose significantly after three days cultivation. These data indicated that the main chemical form of selenium in granulocytes was not GSH-Px. Results from SDS-PAGE revealed a strongly 75Se-labeled protein band with subunit molecular weight of 15 kDa in the supernatant of granulocyte homogenate. However, the main chemical forms of selenium in the culture media of granulocytes and lymphocytes were found to be selenoprotein P. The different forms of selenium-containing proteins in the intracellular and extracellular media of granulocytes indicated the different functions of these proteins.

Characterization of small GTP-Binding proteins in plant cell

To identify and characterize small GTP-binding proteins in plant cells, GTP-binding studies were performed with electroblotted plant proteins following SDS-polyacrylamide gel electrophoresis using [α-32P]GTP. Three species of small GTP-binding protein (21, 23, and 27 kD) which have a specific GTP-binding property were identified in the membrane and cytosolic fractions of both monocotyledons (Zea mays) and dicotyledons (Glycine max). Moreover, these three species of small GTP-binding protein were gradually decreased when membranes were treated with hydroxylamine. This result indicates that these small GTP-binding proteins in plant cells are fatty acylated to the membrane lipids. The 27 kDa component was partially purified from hypocotyl membranes of Glycinemax, following S-300 gel filtration, phenylsepharose CL-4B, hydroxyapatite, and Q-sepharose column chromatography. This 27 kD protein was found to have both GTP-binding and GTPase activities.

Purification and some properties of phospholipase B from Schizosaccharomyces pombe.

Phospholipase B from Schizosaccharomyces pombe was purified by ammonium sulfate fractionation and chromatographed on phenyl-Sepharose CL-4B, DEAE-Toyopearl 650M, and TSK gel G4000SW columns. The purified enzyme was a glycoprotein with molecular weight of approximately 300,000 and 100,000-150,000 by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively. The isoelectric point was pH 4.7. The optimum pH of the enzyme was 2.5 and no activity was detected at neutral and alkaline pHs. The enzyme was not heat-stable. Enzyme activity was slightly stimulated by divalent ions except Fe2+ and 0.1% sodium deoxycholate, and inhibited by Fe2+, Fe3+, 0.1% sodium dodecyl sulfate, and 0.01% cetyltrimethylammonium bromide. The enzyme hydrolyzed mono- and diacylphospholipids, and phosphatidylinositol was hydrolyzed most preferentially. Triglyceride was not hydrolyzed. The enzyme also had acyltransferase activity on lysophosphatidylcholine, forming the corresponding diacylphosphatidylcholine.

Purification and characterization of rat parotid glycosylated, basic and acidic proline-rich proteins.

A unique family of proline-rich proteins (PRPs) is induced in rats following prolonged isoproterenol treatment. PRPs can be divided into glycosylated (GPRP), basic (BPRP) and acidic (APRP) proline-rich proteins based on their physicochemical characteristics. Inducible rat parotid PRPs were isolated from aqueous extracts of parotid glands of isoproterenol-treated animals by sequential chromatography on columns of DEAE-Sepharose CL-6B, Sephadex G-100 and FPLC on Suprose-12 column. The GPRP showed a single homogeneous band on sodium dodecylpolyacrylamide gel electrophoresis with an estimated molecular weight of approximately 220,000. Compositional analysis of GPRP revealed that this protein contained 19.7% glutamic acid/glutamine, 28.2% proline and 9.5% glycine, and 44% carbohydrate, consisting of fucose (2.81g/100g), mannose (9.78g/100g), galactose (9.29g/100g), N-acetylglucosamine (18.03g/100g) and N-acetylgalactosamine (3.90g/100g). Basic PRPs consisted of a family of proteins with estimated molecular masses ranging from 14-45 kDa. These proteins contained 42.6% proline, 20.65% glutamic acid/glutamine and 21.33% glycine. Acidic PRPs also comprised of a family of metachromatically stained ladder of 40-60 kDa containing 29.1% proline, 21.5% glutamic acid/glutamine and 17.8% glycine. APRP were heavily glycosylated containing N-acetylglucosamine (6.34g/100g), N-acetylgalactosamine (19.04g/100g) and glucuronic acid (38.08g/100g).

Purification and N-terminal sequence of a novel interferon-induced 100 kD protein secreted by mouse Ehrlich ascites tumor cells.

Mouse Ehrlich ascites tumor cells secrete a 100 kD protein when they are stimulated by murine interferon-beta (2000 IU/ml). This 100 kD protein was purified from conditioned medium by chromatography on phenyl-Sepharose CL-4B, phosphocellulose, DEAE-Sephacel, and Vydac C4 columns and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single species of 100 kD protein was isolated, and its N-terminal sequence analysis suggested that mouse Ehrlich ascites tumor cell-derived interferon-induced 100 kD protein is a novel protein.

Peculiar properties of lipase fromCandida parapsilosis (Ashford) langeron and talice

A newCandida parapsilosis lipase was isolated and studied. This enzyme was purified by hydrophobic chromatography on a phenyl‐sepharose CL4B column followed by gel permeation on a Sephacryl S300 HR column. It was a 160 kg·mol−1 molecular‐weight oligomeric enzyme. Optimal activity was obtained at 45°C and pH 6.5. The lipase activity toward various acylglycerols and esters was studied. The hydrolysis rate was greater for secondary acylesters than for primary acylesters. This lipase showed a high specificity for long‐chain fatty acids and particularly for polyunsaturated fatty acids. This enzyme was able to catalyze the synthesis of various oleoylesters in aqueous medium.

Purification and characterization of two isoforms of T-kininogens from rat liver microsomes.

T-Kininogen is one of the acute phase proteins, and is a precursor of T-kinin and a cysteine protease inhibitor. Two homologous T-kininogens (TI- and TII-kininogens) were isolated from microsomal fraction of inflamed rat liver, by chromatographies on columns of DEAE-Sepharose CL-6B and DEAE-5PW and by affinity chromatography on a column of anti T-kininogen monoclonal antibody. The amino terminal amino acid sequences of the two microsomal pyridylethylated T-kininogens after pyroglutamyl aminopeptidase treatment were identical with those of TI- and TII-kininogens from inflamed rat plasma. Microsomal T-kininogens moved faster on SDS-PAGE after treatment with endoglycosidase H. The amounts of microsomal TI- and TII-kininogens in inflamed and non-inflamed rat liver were quantitated by immunoblotting of homogenates of liver microsomes using anti T-kininogen rabbit antiserum. The amounts of microsomal T-kininogens were increased in inflamed rat liver, but the ratio of the amounts of TI-kininogen to TII-kininogen was not different in the inflamed and non-inflamed rat liver. On the other hand, TII-kininogen was not significantly detected in non-inflamed rat plasma. These results indicate that the secretion of one of the T-kininogens, TII-kininogen, into plasma may be prevented by some unknown mechanism.

Solubilization and properties of GDP-fucose: Xyloglucan 1,2-α- l-fucosyltransferase from pea epicotyl membranes

GDP-fucose:xyloglucan 1,2-α- l-fucosyltransferase from pea ( Pisum sativum) epicotyl microsomal membranes was readily solubilized by extraction with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps). When using GDP-[ 14C]fucose as fucosyl donor and tamarind xyloglucan (XG) as acceptor, maximum activation was observed at 0.3% ( w v ) Chaps and the highest yield of solubilized activity at 0.4%. The reaction product was hydrolyzed by Trichoderma cellulase to yield labeled oligosaccharides that peaked on gel permeation chromatography at the same elution volume as pea XG nona-and decasaccharide subunits. The apparent K m for fucosyl transfer to tamarind XG by the membrane-bound or solubilized enzyme was about 80 μ m GDP-fucose. This was 10 times the apparent K m for fucosyl transfer to endogenous pea nascent XG. Optimum activity was between pH 6 and 7, and the isoelectric point was close to pH 4.8. The solubilized enzyme showed no requirement for, or stimulation by, added cations or phospholipids, and was stable for several months at −70 °C. Solubilization and gel permeation chromatography on columns of Sepharose CL-6B enriched the specific activity of the enzyme by about 20-fold relative to microsomes. Activity fractionated on columns of CL-6B with an apparent molecular weight of 150 kDa. The solubilized fucosyltransferase was electrophoresed on nondenaturing polyacrylamide slab gels containing 0.02% ( w v ) tamarind XG, and its activity located by incubation in GDP-[ 14C]fucose, washing, and autoradiographing the gel. A single band of labeled reaction product appeared with an apparent molecular weight of 150 kDa.

Histidinoalanine-containing phosphoprotein in bone

Histidinoalanine is a naturally occurring cross-linking amino acid found in mammalian bone, dentin, cartilage and aorta, and also in the extracellular fluid of bivalve clams. The exact components from which this amino acid derives have not been characterized in the case of bone. In this study, the origin of histidinoalanine in bone was investigated in order to clarify the possible role of the matrix component containing this amino acid in bone mineralization. An extract of bovine bone powder with 0.5 M EDTA/1M NaCl was desalted and separated by calcium-induced precipitation. The supernatant fraction was chromatographed successively on Sepharose CL-6B, hydroxyapatite, DEAE cellulose, and reverse-phase high-performance liquid chromatography columns. The fraction with the highest affinity for hydroxyapatite was found to contain the highest concentration of histidinoalanine. A phosphoprotein with a molecular weight of about 24 K that contained 1.2 mol of histidinoalanine per molecule was isolated. The amino acid composition of this fraction showed a high content of acidic amino acids and serine, at least 42% of which was phosphorylated. These results suggested a possible formation mechanism of histidinoalanine that includes the beta-elimination of phosphoserine, producing a dehydroalanine residue and an addition of histidine on it. It was concluded that this low molecular weight phosphoprotein is one of the major sources of histidinoalanine in bovine bone.

Axonal transport of proteoglycans to the goldfish optic tectum.

The study addressed the question of whether 35SO4 labeled molecules that have been delivered to the goldfish optic nerve terminals by rapid axonal transport include soluble proteoglycans. For analysis, tectal homogenates were subfractionated into a soluble fraction (soluble after centrifugation at 105,000 g), a lysis fraction (soluble after treatment with hypotonic buffer followed by centrifugation at 105,000 g) and a final 105,000 g pellet fraction. The soluble fraction contained 25.7% of incorporated radioactivity and upon DEAE chromatography was resolved into a fraction of sulfated glycoproteins eluting at 0-0.32 M NaCl and containing 39.5% of total soluble label and a fraction eluting at 0.32-0.60 M NaCl containing 53.9% of soluble label. This latter fraction was included on columns of Sepharose CL-6B with or without 4 M guanidine and after pronase digestion was found to have 51% of its radioactivity contained in the glycosaminoglycans (GAGs) heparan sulfate and chondroitin (4 or 6) sulfate in the ratio of 70% to 30%. Mobility of both intact proteoglycans and constituent GAGs on Sepharose CL-6B indicated a size distribution that is smaller than has been observed for proteoglycans and GAGs from cultured neuronal cell lines. Similar analysis of lysis fraction, containing 11.5% of incorporated 35SO4, showed a mixture of heparan sulfate and chondroitin sulfate containing proteoglycans, apparent free heparan sulfate and few, if any, sulfated glycoproteins. Overall, the results support the hypothesis that soluble proteoglycans are among the molecules axonally transported in the visual system.

Immunologically active polysaccharides of acanthopanax senticosus

From the alkaline aqueous extract of Acanthopanax (Eleutherococcus) senticosus two homogeneous polysaccharides, a glucan with a mean M, of 150 000 and a heteroxylan with a mean M, of 30 000, have been isolated by DEAE-Sepharose CL-6B and Sephacryl S-400 column chromatography. Their structures were elucidated mainly by permethylation, periodate oxidation, Smith degradation, reduction experiments, partial hydrolysis and 13C NMR. The isolated crude polysaccharide mixture and the heteroxylan enhance phagocytosis in in vitro and in vivo immunological tests.

Purification of [formula omitted] lysing yeast cell walls from Rhizoctonia solani

Three forms of endo-(1→3)-β- g-glucanases lysing yeast cell walls from Rhizoctonia solani were separated by precipitation with ammonium sulfate and by successive chromatographies on CM Bio-Gel A and Bio-Gel P-60 or P-30, and were finally purified by substrate affinity chromatography on short-chain pachyman-AH-Sepharose CL 6B column. Each preparation was found to be homogeneous on gel filtration and by electrophoresis on acrylamide gel with sodium dodecyl sulfate. They exhibit high activity against insoluble pachyman, but only restricted activity against soluble short-chain pachyman. In the affinity chromatography, three enzymes were found to be strongly absorbed on the column, so that they could be easily eluted with substrate solution using biospecific counter-ligand. It was thus revealed that covalent binding of such a soluble glucan to aminohexyl-Sepharose provides a useful carrier for separation of endo-(1→3)-β- D-glucanases lysing yeast cell walls.

Glycoprotein Allergens in Pollen of Timothy

A basic glycoprotein allergen has been isolated from pollen of timothy by a combination of chromatography on columns of CM-Sepharose CL-6B, Bio-Gel P-30, DEAE-Sepharose CL-6B and Ultrogel AcA 44. The allergen which is immunologically identical with the allergen formerly denoted as antigen 30 had an isoelectric point of 9.45. The MW of the glycoprotein was found to be 54,000 and 38,000 by SDS-PAGE and gel filtration, respectively. The allergen contains arabinose (3.1%), fucose (0.7%), xylose (0.7%), mannose (2.1%), galactose (2.8%) and glucose (0.2%). In rocket radioimmunoelectrophoresis the allergen bound IgE from 57 (78%) of 73 patient sera tested. The allergen did not bind Yariv artificial galactosyl- and glucosyl-carbohydrate-binding antigens or a monoclonal antibody against ryegrass pollen glycoprotein allergens.

Purification and properties of a DNA ligase from sea urchin embryos.

DNA ligase was purified about 2,000-fold from blastulae of sea urchin, Hemicentrotus pulcherrimus, by means of 1 M KCl-extraction, phosphocellulose, DEAE-cellulose, Sepharose CL-6B, and double-stranded DNA cellulose column chromatography. The purified DNA ligase had a molecular weight of 80,000 (determined by Sephadex G-150) and a sedimentation coefficient of 4.1S (by glycerol gradient centrifugation). The purified enzyme required ATP and Mg2+ (or Mn2+) as cofactors for activity, and was inhibited by N-ethylmaleimide. Apparent Km values for ATP, Mg2+, and Mn2+ were 4 microM, 2.7 mM, and 0.3 mM, respectively.

Solubilization and partial characterization of a tumor-rejection antigen from an ultraviolet light-induced murine tumor.

Tumor rejection antigen (TRA) of an ultraviolet-light-induced murine skin tumor was solubilized, fractionated and partially characterized. Subcellular fractions were prepared by differential centrifugation of tumor cells that had been ruptured via nitrogen cavitation. Only the 110,000-g membrane fraction induced significant tumor protection, as determined by in vivo immunization and challenge assays. Extraction of the membrane fraction with 3 M KCI resulted in solubilization of material that could induce in vivo tumor-rejection immunity. Both the membrane fraction and soluble extract had a limited effective dose range. The KCI extract was separated on a Sepharose, CL-6B column in the presence of 6 M guanidine-HCI. Only one of five fraction pools (molecular weight range of 76,000-127,000 daltons) was immunogenic. It contained at least eight protein bands by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), but no lipid components. This immunogenic Sepharose fraction was chromatographed on a Sephadex G-150 column. Each of the four Sephadex fraction pools was immunogenic. One protein component was common to each of those fractions. It migrated as a single 76,000-dalton band on SDS-PAGE and contained [14C]-leucine and [3H]-glucosamine that had been incorporated during cell growth. These results suggest that the TRA of this tumor is a 76,000-dalton glycoprotein.

Separation of protein-bound copper and zinc in human plasma by means of gel filtration -ion-exchange chromatography.

A procedure for the separation of protein-bound copper and zinc fractions and of potassium and magnesium in plasma, involving chromatography on columns of DEAE-Sepharose CL-6B, is described. Copper, magnesium and zinc were measured by atomic-absorption spectroscopy and potassium by flame photometry. Most plasma samples yielded one copper and two zinc fractions and these were identified by the use of protein “markers.” Most of the copper appeared to be bound to caeruloplasmin and a lesser amount to albumin, whereas zinc appeared to be bound mainly to globulins with a smaller, variable, amount bound to albumin. Magnesium and potassium appeared as single peaks eluting in approximately the same fractions, ahead of the albumin peak.