Effect of beef broth protein on the thermal inactivation of staphylococcal enterotoxin B1.

Enterotoxin B produced by Staphylococus aureus 243 in brain heart infusion broth was concentrated by dialysis against 40% polyethylene glycol (20 M), partially purified on a Sephadex G-100 column and heated at 110 degrees C in thermal death time cans. Various heating menstrua included 0.04 M Veronal buffer (pH 7.4), beef broth, and fractions of beef broth obtained by ultrafiltration or precipitation with ammonium sulfate. The toxin was assayed serologically using the microslide gel double-diffusion method. The time requiring for 90% inactivation at 110 degrees C (D110 value) obtained in buffer and in beef broth was 18 and 60 min, respectively. When the concentration of beef broth was increased fivefold, the D110 increased to 78 min. The apparent protective effect or protein was further investigated using beef broth protein obtained by precipitation with (NH4)2SO4. The D110 values were 51 and 70 min when the protein concentration in the heating menstruum was 3.8 and 7.7 mg/ml, respectively. However, when the beef broth protein was dialyzed against buffer before use as a heating menstrum, the D110 was only 39 or 41 min at comparable protein concentrations. Results indicated a dialyzable factor, whose protective effect was partially destroyed by trypsin and chymotrypsin but did not by disodium ethylenediaminetetraacetate, was involved in the protection of enterotoxin B during heating.     

Effect of beef broth protein on the thermal inactivation of staphylococcal enterotoxin B1.

Enterotoxin B produced by Staphylococus aureus 243 in brain heart infusion broth was concentrated by dialysis against 40% polyethylene glycol (20 M), partially purified on a Sephadex G-100 column and heated at 110 degrees C in thermal death time cans. Various heating menstrua included 0.04 M Veronal buffer (pH 7.4), beef broth, and fractions of beef broth obtained by ultrafiltration or precipitation with ammonium sulfate. The toxin was assayed serologically using the microslide gel double-diffusion method. The time requiring for 90% inactivation at 110 degrees C (D110 value) obtained in buffer and in beef broth was 18 and 60 min, respectively. When the concentration of beef broth was increased fivefold, the D110 increased to 78 min. The apparent protective effect or protein was further investigated using beef broth protein obtained by precipitation with (NH4)2SO4. The D110 values were 51 and 70 min when the protein concentration in the heating menstruum was 3.8 and 7.7 mg/ml, respectively. However, when the beef broth protein was dialyzed against buffer before use as a heating menstrum, the D110 was only 39 or 41 min at comparable protein concentrations. Results indicated a dialyzable factor, whose protective effect was partially destroyed by trypsin and chymotrypsin but did not by disodium ethylenediaminetetraacetate, was involved in the protection of enterotoxin B during heating.     

Tetanus Toxin and Antigenic Derivatives I. Purification of the Biologically Active Monomer

A procedure for the isolation of pure tetanus toxin in a lethal monomeric form was developed based on the extraction of whole cells and chromatographic techniques. A crude extract of toxin was obtained by hypertonic extraction of cells from a 72-hr culture of Clostridium tetani Massachusetts strain. The extract was precipitated with ammonium sulfate and further purified by sequential use of ion-exchange chromatography and gel filtration. The degree of purification obtained by the fractionation procedures was monitored by polyacrylamide gel electrophoresis. The pure toxin has an average specific activity of 150 x 10(6) mouse MLD per mg of N and 3,000 Lf per mg of N. Immunological purity was demonstrated by a single line on both immunoelectrophoresis and agar double diffusion. One band was obtained on polyacrylamide electrophoresis, as was a single symmetrical peak in the ultracentrifuge and on Sephadex G-100 chromatography. The pure protein has an absorbancy ratio (280/260 mmu) of 2.1 in phosphate buffer (pH 7.5).     

Purification and characterisation of tyrosine decarboxylase and aromatic-L-amino-acid decarboxylase

Microbial tyrosine decarboxylase (EC 4.1.1.25) and mammalian aromatic-L-amino-acid decarboxylase (EC 4.1.1.28) catalyse the formation of tyramine from L-tyrosine. These enzymes were characterised after isolation to purity by methods including fast polymer liquid chromatography (FPLC). Tyrosine decarboxylase was isolated from Streptococcus faecalis by FPLC anion exchange chromatography (11-times purification; 72% recovery; 23.2 U/mg protein). FPLC on Phenyl-Superose resulted in purification to 115 U/mg protein. Aromatic-L-amino-acid decarboxylase was isolated from pig kidney by ammonium sulfate fractionation, DEAE chromatography, and FPLC anion exchange chromatography (21-times purification; 22% recovery; 0.71 U/mg protein). By FPLC chromatofocusing, tyrosine decarboxylase eluted at pH 4.3 and aromatic-L-amino-acid decarboxylase at pH 5.0. Isoelectric focusing of tyrosine decarboxylase gave two bands (pI 4.4 and 4.5). With pyridoxal 5'-phosphate removed by ultrafiltration, only one band (pI 4.4) appeared, and SDS polyacrylamide electrophoresis confirmed the purity. FPLC gel filtration resulted in molecular weights 143,000 and 86,000, respectively, for tyrosine decarboxylase and aromatic-L-amino-acid decarboxylase. In SDS electrophoresis, tyrosine decarboxylase had the monomer molecular weight 75,000, showing a dimer structure for the enzyme.     

Strategy for a protein purification design using C-phycocyanin extract

A variety of techniques have been developed for the separation and recovery of proteins. The cost of purifying the product is frequently determined by the desired quality of the final product, which is evaluated by measuring the purity. In this work the design of a protein purification process for C-phycocyanin, a phycobiliprotein that can be used in the food and medical industries, was established. The study evaluated the use of ammonium sulfate precipitation, ion exchange chromatography and gel filtration to purify C-phycocyanin in a variety of sequences. The final design included the C-phycocyanin extraction step, precipitation with ammonium sulfate and ion exchange chromatography. When the elution step was studied, the kind of elution and pH were considered in order to obtain a product with a final purity of 4.0 with a purification factor of 6.35, so that, at the end of the strategy, C-phycocyanin of analytical grade would be obtained.     

Degradative acetolactate synthase of Bacillus subtilis: purification and properties.

A degradative acetolactate synthase (acetolactate pyruvate-lyase [carboxylating], EC 4.1.3.18) from Bacillus subtilis has been partially purified and characterized. The synthesis of the enzyme was induced by growth of cells in minimal medium plus isobutyrate or acetate. The enzyme was partially purified by ammonium sulfate fractionation, gel filtration, and hydroxyapatite chromatography. The pH optimum of the purified enzyme was 7.0 in phosphate buffer. When assayed in phosphate buffer (pH 7.0), activity was stimulated by acetate and inhibited by sulfate. When assayed in acetate buffer (pH 5.8), activity was inhibited both by sulfate and phosphate. Michaelis-Menten kinetics was observed when the enzyme was assayed in phosphate buffer (pH 6.0 or 7.0), and inhibition by sulfate was competitive and activation by acetate was noncompetitive. When assayed in acetate buffer (pH 5.8), nonlinear Lineweaver-Burk plots were obtained; inhibition by phosphate appeared to be competitive and that by sulfate was of the mixed type. The approximate molecular weight of the purified enzyme was 250,000 as determined by gel filtration.     

Membrane ultrafiltration: concentration of interleukin-3.

A pilot-plant-scale operation was used for studying membrane ultrafiltration and concentration of kiloliter quantities of the lymphokine interleukin-3 with a single set of membranes. Initial use of ammonium sulfate precipitation of interleukin-3 proved erratic in the recovery of biological activity and resulted in corrosion of the processing equipment. Membrane ultrafiltration proved to be effective in enabling control of the degree of concentration and predicting recovery of the biologically active protein.     

Selection of method for obtaining an active mutanase preparation from Trichoderma harzianum

Crude mutanase preparations of Trichoderma harzianum were obtained from the culture supernatant by means of ammonium sulfate salting out, ultrafiltration, freeze-drying, concentration under reduced pressure, and fractional precipitation with organic solvents (methanol, ethanol, propanol, isopropanol, acetone). Ammonium sulfate was the worst precipitant, causing a fall in total mutanase activity by 47%. Other methods of enzyme recovery from the post-culture fluid yielded in most cases very good results in regard to specific and overall activities of the enzymatic preparations.     

Resolution and reconstitution of succinate-cytochrome c reductase: preparations and properties of high purity succinate dehydrogenase and ubiquinol-cytochrome c reductase

An improved method was developed to sequentially fractionate succinate-cytochrome c reductase into three reconstitutive active enzyme systems with good yield: pure succinate dehydrogenase, ubiquinone-binding protein fraction and a highly purified ubiquinol-cytochrome c reductase (cytochrome b-c1 III complex). An extensively dialyzed succinate-cytochrome c reductase was first separated into a succinae dehydrogenase fraction and the cytochrome b-c1 complex by alkali treatment. The resulting succinate dehydrogenase fraction was further purified to homogeneity by the treatment of butanol, calcium phosphate gel adsorption and ammonium sulfate fractionation under anaerobic condition in the presence of succinate and dithiothreitol. The cytochrome b-c1 complex was separated into chtochrome b-c1 III complex and ubiquinone-binding protein fractions by careful ammonium acetate fractionation in the presence of deoxycholate. The purified succinate dehydrogenase contained only two polypeptides with molecular weights of 70 000 anbd 27 000 as revealed by the sodium dodecyl sulfate polyacrylamide gel electrophoretic pattern. The enzyme has the reconstitutive activity and a low Km ferricyanide reductase activity of 85 mumol succinate oxidized per min per mg protein at 38 degrees C. Chemical composition analysis of cytochrome b-c1 III complex showed that the preparation was completely free of contamination of succinate dehydrogenase and ubiquinone-binding protein and was 30% more pure than the available preparation. When these three components were mixed in a proper ratio, a thenoyltrifluoroacetone- and antimycin A-sensitive succinate-cytochrome c reductase was reconstituted.     

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.     

Rule of antibody structure: the primary structure of a human monoclonal IgA1-immunoglobulin (myeloma protein Tro), I. Purification and characterization of the protein and its L- and H-chains (author's transl)]

Myeloma protein Tro was prepared from the serum of a myeloma patient by ammonium sulfate precipitation. It was purified by gel filtration and ion exchange chromatography or by Pevikon block electrophoresis. The purity of the preparation was tested by several electrophoretic or immunoelectrophoretic methods. L- and H-chains of the purified protein after reduction and alkylation were separated by gel chromatography. The protein and its L- and H-chains were characterized by amino acid analyses and end-group determinations.     

Purification of penicillin acylase of Bacillus megaterium

Penicillin acylase from Bacillus megaterium was purified 6.22 fold using ultrafiltration followed by ammonium sulphate precipitation and gel filtration chromatography using Sephadex G-100. The final specific activity was 2.62 U/mg protein and was free from interfering proteases.     

Evaluation of sample extraction methods for proteomic analysis of coniferous seeds

In this study, three methods of protein extraction from the seeds of the Chinese fir were compared by examining the quality (including the number of protein spots observed) in the two-dimensional gel electrophoresis (2-DE), obtained by isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis of the total released protein. Three protein extraction methods were: TCA-acetone precipitation, SDS extraction/acetone precipitation, and phenol extraction methanol/ammonium acetate precipitation. The results showed that TCA-acetone precipitation was the most effective method for protein extraction; it gave the highest yield of total protein (8.9 mg protein per g seed weight) and the greatest number of proteins spots (1,034 spots) on the 2-DE gel. Further, several proteins were identified by liquid chromatography mass spectrometry (LC MS/MS), which are legumin-like storage protein, similar to AMP binding/acetate-CoA ligase, similar to 40S ribosomal protein S20, actin, ascorbate peroxidase, Similar to cysteine synthase, and unknown protein. These data demonstrates that TCA-acetone precipitation followed by 2-DE and LC MS/MS is a suitable method for proteomic analysis of coniferous species, such as Chinese fir and provides a valuable starting point for similar proteomic analysis of other coniferous tree species.     

Efficient recovery of recombinant human erythropoietin from milk of transgenic pigs by two-step pretreatment

The selective removal of impurity proteins and colloidal particles from milk prior to chromatographic purification processes presents a crucial issue in the production of therapeutic proteins from transgenic animals with high recovery yield and purity. We have developed an efficient two-step precipitation method for the recovery of the recombinant human erythropoietin (rhEPO) of interest from transgenic sow milk. Here, rhEPO was partially purified from transgenic sow milk via a two-step precipitation method consisting of ammonium sulfate and divalent metal precipitations, with a yield of approximately 82.1% and a purification fold of 10.4 at a copper concentration of 30 mM. Copper proved to be the strongest flocculating agent among the divalent ions tested for the aggregation of milk proteins under 35%, with ammonium sulfate, zinc, nickel, and calcium demonstrating increasing flocculating capability in the given order. Copper and zinc proved to be appropriate divalent metals for the recovery of rhEPO at high yield and purity, and the optimal concentration ranges of copper and zinc were 20~40 and 40~80 mM, respectively.     

Purification and immobilization of acetate kinase from Desulfovibrio vulgaris

Summary The enzyme acetate kinase (EC 2.7.2.1) was purified fromDesulfovibrio vulgaris by a combination of ammonium sulfate precipitation, hydroxyl-apatite and dye-affinity chromatography. An overall-purification factor of 15 was obtained resulting in a specific activity of 24 U/mg protein. The purified enzyme was immobilized on differently derivatized controlled pore glass beads.     

Partial purification and characterization of extracellular cellulase from a strain ofTrichoderma viride isolated from forest soil

Partial purification of extracellular cellulase ofTrichoderma viride isolated from forest soil was done by ammonium sulfate precipitation of culture supernatant, centrifugation at higher speed, solubilization of protein in sodium acetate buffer and dialysis. The specific activity of cellulase in the culture supernatant, was 136 nkat/mg which was increased by 172% after the completion of final step (234 nkat/mg). The recovery of enzyme was 70%. The enzyme was characterized by demonstration of optimum activity at 55°C and pH 5.0 with 1% carboxymethyl cellulose as substrate.     

Pyruvate kinase from human skeletal muscle

A simple method is described for the isolation of crystalline pyruvate kinase from human skeletal muscle. The enzyme was purified by ammonium sulfate fractionation, heat treatment and crystallization. Two crystal forms of pyruvate kinase differing in solubility but not in specific activity were found. The homogenous enzyme preparations in triethanolamine buffer, pH 7.6 reveal at 25 degrees a specific activity of 245 U per mg protein, and of 340 U/mg in potassium phosphate buffer (50 mM). The enzyme is activated by inorganic phosphate and fructosediphosphate to the same extent, and inhibited non competetively by ammonium ion. The molecular weight as measured by gel filtration is 220,000 daltons and the enzyme molecule is composed of 4 subunits.     

赤子爱胜蚓超氧化物歧化酶的纯化和部分性质研究

采用硫酸铵分级沉淀和柱层析的方法,从赤子爱胜蚓整体细胞抽提液内分离得到纯的铜锌超氧化物岐化酶（Ｃｕ,Ｚｎ－ＳＯＤ）。每１００ｇ蚯蚓得到的ＳＯＤ制品,总活力为１１１５０Ｕ,比活力为５１３８Ｕ／ｍｇ蛋白,回收率为２０％。铜锌超氧化物岐化酶呈淡蓝绿色,最大紫外吸收波长为２７０ｎｍ。测得该酶分子量为３３０００,亚基分子量为１６５００。该酶亚基由１５６个氨基酸残基组成,不含酪氨酸。