Purification and Properties of Clostridium botulinum Type F Toxin

Clostridium botulinum type F toxin of proteolytic Langeland strain was purified. Toxin in whole cultures was precipitated with (NH₄)₂SO₄. Extract of the precipitate was successively chromatographed on diethylaminoethyl-cellulose at pH 6.0, O-(carboxymethyl) cellulose at pH 4.9, Sephadex G-200 at pH 8.1, quaternary aminoethyl-Sephadex at pH 4.9, and finally diethylaminoethyl-cellulose at pH 8.1. The procedure recovered 14% of the toxin assayed in the starting culture. The toxin was homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, double gel diffusion serology, and isoelectric focusing. Purified toxin had a molecular weight of 150,000 by gel filtration and 155,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Specific toxicity was 9.6 × 10⁶ mean lethal doses per absorbancy (278 nm) unit. Sub-units of 105,000 and 56,000 molecular weight are found when purified toxin is treated with a disulfide reducing agent and electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. Reciprocal cross neutralizations were demonstrated when purified type F and E toxins were reacted with antitoxins which were obtained with immunizing toxoids prepared with purified toxins.     

Removal of Adsorbed Toxin Fragments That Modify Bacillus thuringiensis CryIC δ-Endotoxin Iodination and Binding by Sodium Dodecyl Sulfate Treatment and Renaturation

We report that 10- and 25-kDa toxin fragments adhere to CryIC prepared from Bacillus thuringiensis insecticidal crystals, block iodination, and alter membrane binding. There is no apparent affect on CryIC toxicity against Spodoptera exigua. Associated peptides remained bound to CryIC in the presence of 50 mM dithiothreitol or 6 M urea. A novel detergent-renaturation procedure was developed for the purification of B. thuringiensis CryIC toxin. Sodium dodecyl sulfate (SDS) treatment followed by gel filtration chromatography yielded a homogeneous 62-kDa CryIC toxin. After removal of SDS and renaturation, the purified CryIC toxin was fully insecticidal to S. exigua larvae. ¹²⁵I-labeled CryIC bound with high affinity to brush border membrane vesicles from S. exigua larvae.     

Isolation and Biological Activities of Four Selective Toxins from Helminthosporium carbonum

A new and simpler purification procedure was developed for host selective toxins from Helminthosporium carbonum race 1. Four analogs or forms of toxin with the same selectivity as the fungus were isolated from culture fluids; two forms (HC toxins III and IV) have not been reported by other workers. Crystals of the major form of toxin (HC toxin I) were recovered in high yields (>80 milligrams per liter of culture fluid) without the use of high performance or preparative thin layer liquid chromatography. ED₅₀ values, based on inhibition of root growth of susceptible seedlings, for HC toxins I, II, III, and IV were 0.2, 0.4, 2.0, and 20 micrograms per milliliter, respectively. The specific activity of crystalline HC toxin I matched the most active preparation reported previously; the preparation of HC toxin II was more active than that reported previously. Resistant seedlings tolerated 100-fold higher concentrations of each form of toxin than did susceptible seedlings. Hydrolysis of the epoxide group of HC toxin I to a diol destroyed toxicity to susceptible and resistant seedlings. The data suggest that the same mechanisms are affected in resistant and susceptible plants.     

Production and purification ofStaphylococcus aureus toxic-shock toxin

Staphylococcus aureus strain 5761, isolated from a patient with toxic-shock syndrome, was used for the production of toxic-shock toxin. The medium used contained 4% bio-Trypcase and 1% yeast extract adjusted to pH 7. Production of 50 μg of toxic-shock toxin/ml of culture supernatant was obtained. The purification method involves removal of the toxin from the culture supernatant with Biorex 70 resin and purification by isoelectric focusing, on 2% (pH 3–10) ampholine-sucrose gradients, and gel filtration on Sephadex G-100. Three antigenically similar entities were isolated after electrofocusing, with a major component at isoionic point pH 7.4. The purified toxin migrated as a homogeneous protein with a molecular weight of 23,700 when tested by gel electrophoresis. Specific antibodies to toxic-shock toxin in rabbits were obtained after one subcutaneous injection of 5 μg enterotoxin.     

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 CRYSTALLIZATION OF DIPHTHERIA ANTITOXIN

Purified preparations of diphtheria antitoxin have been obtained by digestion of the toxin-antitoxin complex with trypsin, followed by fractional precipitation with ammonium sulfate. The various fractions obtained in this way are all 90 per cent or more precipitated by diphtheria toxin but combine with different quantities of the toxin. The fraction precipitated between 0.33 and 0.5 saturated ammonium sulfate is homogeneous by electrophoresis and ultracentrifuge but does not have constant solubility. A small amount of a more soluble fraction has been obtained which does have constant solubility and satisfies the criteria of a pure protein. This protein crystallizes readily in poorly formed thin plates. It is very unstable and reverts to a less soluble non-crystallizable form. It has a sedimentation constant of 5.7 x 10^–13 and a molecular weight of 90,500. It has an antitoxic value of 700–900 flocculation units per mg. protein nitrogen and has an antitoxic value by the protection test of about 700 units per mg. protein nitrogen. The precipitation range of the purified antitoxin with purified toxin is much wider than that with crude preparations.     

The Toxins of Helminthosporium maydis (Race T) : A Colorimetric Determination of the Toxins, Their Appearance in Culture and in Infected Plants

Host-specific toxins produced by Helminthosporium maydis, race T, are measured quantitatively by a chemical assay procedure involving reaction of the toxins with a sulfuric acidacetic anhydride reagent and measurement of the absorbance of the product at 330 nm. The assay was shown to measure total toxin concentrations after only limited fractionation of the culture medium. Using the assay it was possible to show that the highest amount of toxin per gram of fungus mycelium occurs early in the growth cycle of H. maydis. Toxins I, II, and V are the predominant toxins at these early times both in culture and in infected corn and wheat varieties. Some chromatographic and spectral properties of toxin V, a previously unreported toxin, are described. Since toxin V appears in culture prior to toxins I, II, III and IV, a precursor-product relationship can be suggested.     

The purification of hydrogenase II (uptake hydrogenase) from the anaerobic N2-fixing bacterium Clostridium pasteurianum

The H₂ uptake activity (units/mg protein) of Clostridium pasteurianum cells with methylene blue as the electron acceptor increases with cell density independent of the growth conditions. The H₂ evolution activity (units/mg protein) of the same cells with reduced methyl viologen as the electron donor remains fairly constant under all growth conditions tested. Cells grown under N₂-fixing conditions have the highest H₂ uptake activity and were used for the purification of hydrogenase II (uptake hydrogenase). Attempts to separate hydrogenase II from hydrogenase I (bidirectional hydrogenase) by a previously published method were unreliable. We report here a new large-scale purification procedure which employs a rapid membrane filtration system to fractionate cell-free extracts. Hydrogenases I and II were easily filtered into the low-molecular-weight fraction (M_r less than 100 000), and from this, hydrogenase II was further purified to a homogeneous state. Hydrogenase II is a monomeric iron-sulfur protein of molecular weight 53 000 containing eight iron atoms and eight acid-labile sulfur atoms per molecule. Hydrogenase II catalyzes both H₂ oxidation and H₂ evolution at rates of 3000 and 5.9 μmol H₂ consumed or evolved/min per mg protein, respectively. The purification procedure for hydrogenase II using the filtration system described greatly facilitates the large-scale purification of hydrogenase I and other enzymes from cell-free extracts of C. pasteurianum.     

Isolation and Some Properties of the Enzyme That Transforms Eremofortin C to PR Toxin

PR toxin and eremofortin C are secondary metabolites of Penicillium roqueforti. The chemical structures of these two compounds are closely related to each other and differ only by an aldehyde and an alcohol group at the C-12 position. In an effort to better understand the biosynthesis of PR toxin, we discovered the enzyme of P. roqueforti that is responsible for the transformation of eremofortin C to PR toxin. The maximum activity of the enzyme in the culture medium was found to occur on day 13, which corresponded to the maximal production of PR toxin in the medium. The enzyme was isolated and purified from the culture medium and the mycelium of the fungus, respectively, through a procedure involving ammonium sulfate fractionation and DEAE-cellulose chromatography. The specific activity increased 20- and 8-fold, respectively, and the yield was 33.3 and 21.6%, respectively, for the enzyme from the medium and mycelium. The optimal pH for the enzyme reaction was ca. pH 5.6. The enzyme reaction was temperature dependent. The rates followed a linear time course when it catalyzed the transformation at 30°C and decayed with time when reacted at higher temperatures. At 100°C, the enzyme activity was completely lost. The K_m and V_max of the enzyme as determined at 30°C were 0.02 mM and 4.0 μmol/min per mg, respectively. The molecular weight of the enzyme was estimated by gel filtration on a high-pressure liquid chromatography I-250 protein column to be ca. 40,000.     

Introduction of Additional Charges as an Aid in Protein Purification: Isolation of Elongation Factor 2 from Sulfolobus acidocaldarius by Preparative Isoelectric Focusing Before and After ADP-Ribosylation

Diphtheria toxin catalyzes the ADP-ribosylation of elongation factor 2 (EF-2) in eukaryotes and archaebacteria. As the reaction is strictly EF-2 specific and introduces two negative charges into the molecule, the resulting shift in the isoelectric point (pI) by 0.2 pH units was used to establish a new purification method for EF-2 from Sulfolobus acidocaldarius. The cells were lysed with dithiothreitol at pH 9 and EF-2 was purified by ammonium sulfate precipitation, gel filtration on Sephadex G-200, and three isoelectric focusing steps. The EF-2-containing fractions from the first isoelectric focusing step at pH 4-9 were refocused in a more narrow pH-gradient (pH 5-7). The EF-2 peak from the second step was eluted, collecting only the fractions above the pH region where ADP-ribosylated EF-2 would focus. The EF-2 was then ADP-ribosylated with diphtheria toxin and NAD and subjected to further isoelectric focusing (pH 5-7). The EF-2 was almost homogeneous since ADP-ribosylation had shifted it into a region of the pH gradient free of contaminating proteins. Diphtheria toxin was immobilized on CNBr-activated Sepharose to prevent a possible contamination by proteins from the diphtheria toxin preparation which might have the same pI as ADP-ribosylated EF-2. Finally, the ADP-ribosyl group was removed by equilibrium dialysis using diphtheria toxin and nicotinamide at pH 6.3. The obtained EF-2 was active in protein synthesis.     

Purification and characterization of streptococcal erythrogenic toxin type A produced by Streptococcus pyogenes strain NY-5 cultured in the synthetic medium NCTC-135. Comparison with the dialyzed medium (TP medium)-derived toxin

Streptococcal erythrogenic toxin type A (ET-A) was purified from culture filtrate of Streptococcus pyogenes strain NY-5 grown in a chemically defined synthetic medium NCTC-135. We succeeded in simplifying the purification procedure, and obtained a highly purified preparation of ET-A. The purification procedure was the combination of ultrafiltration with Amicon PM-10 and YM-10 membranes, chromatofocusing with PBE-94 exchanger (pH 4.0-6.0), and gel filtration through Sephacryl S-200. The purified toxin protein showed a single band with Mr 28,000 on SDS-PAGE and had pI 5.2 on agarose IEF. HPLC chromatography pattern of the toxin revealed one symmetric peak. The result of amino acid analysis of the toxin was in accordance with that of Gerlach et al and with Weeks and Ferretti who reported the nucleotide sequence of the spe A gene. Biological activities of the purified toxin were remarkably potent. The mitogenic activity for rabbit lymphocytes and one skin test dose in rabbit were found at the lower dose of 10 pg and 1 ng of the toxin, respectively.     

Hemorrhagic toxin from the venom of Agkistrodon bilineatus (common cantil)

1. Hemorrhagic toxin was isolated from Agkistrodon bilineatus (Common cantil) venom using a three-step purification procedure to obtain 32.8 mg of purified hemorrhagic toxin from 700 mg of crude venom. 2. The purified toxin was homogeneous by disc polyacrylamide gel electrophoresis at pH 8.3, and by isoelectric focusing. 3. Hemorrhagic toxin possessed lethal, hemorrhagic and proteolytic activities. These activities of this toxin were inhibited by ethylenediaminetetraacetic acid (EDTA) and ethyleneglycol-bis-(beta-aminoethylether)N,N'-tetraacetic acid (EGTA), but not by cysteine or soybean trypsin inhibitor (SBTI). 4. Its molecular weight was approximately 48 kDa and the isoelectric point was 4.2. 5. Purified preparation hydrolyzed the Asn(3)--Gln(4), His(10)--Leu(11), Ala(14)--Leu(15), Tyr(16)--Leu(17), Arg(22)--Gly(23) and Phe(24)--Phe(25) bonds of oxidized insulin B. chain. 6. The A alpha chain of fibrinogen was first split and B beta chain was cleaved later by this toxin. 7. Hemorrhagic toxin contains 1 mol of zinc and 2 mol of calcium per mol of protein.     

Human tissue-type plasminogen activator. Production in continuous serum-free cell culture and rapid purification.

A simplified procedure for the production and purification of human tissue-type plasminogen activator (t-PA) is described. Bowes-melanoma cells were maintained in continuous serum-free culture. The cell nutrient consisted of Dulbecco's modified Eagle's medium (DMEM) supplemented with insulin (5 mg/litre), transferrin (5 mg/litre), progesterone (1 nM), cortisol (10 nM), aprotinin (2 X 10(4) units/litre) and a mixture of trace elements. t-PA accumulated in the culture medium at a rate of 40 units/day per ml and was harvested every third day. Cell losses during each harvest, leading to a steady decline of enzyme yields, were compensated for by treating the cells with 5% (v/v) fetal-bovine serum in DMEM every 6-8 weeks. t-PA was rapidly purified by a combination of cation-exchange chromatography and gel filtration. The procedure yielded mainly single-chain t-PA of a specific activity of 80 000 to 100 000 units/mg.     

Selective Toxins and Analogs Produced by Helminthosporium sacchari: Production, Characterization, and Biological Activity

Helminthosporium sacchari toxin and several lower molecular weight, nontoxic analogs were isolated from culture filtrates. Three isomers of the toxin (A, B, and C), each with four galactose units, were separated by high performance liquid chromatography. Isomer C had the highest and isomer A had the lowest toxicity to H. sacchari-susceptible sugarcane; resistant clones were not affected. Each toxin isomer was partially digested with a β-galactofuranosidase and the resulting analogs (seven from each toxin isomer) were separated by reverse phase high performance liquid chromatography and identified. Each isomer of the analogs with 3 galactose units per mole also was partially digested and the arrangement of galactose units was determined. The compound with one galactose attached to position 2 of the bicyclic sesquiterpene and with 2 galactose units attached to position 13 (analog A_1,2) was highly toxic to some but not to all clones of H. sacchari-susceptible sugarcane. Toxin analogs protected sensitive tissue against active toxin; protective effects of the analogs differed, but at least a 10-fold excess of analog was required. Analog C_2,1 was more effective at preventing toxin C-induced electrolyte losses than was any other analog. Each of the 3-galactose analog isomers protected better than did any of the 2-galactose compounds. The 1,1 analogs did not protect as well as did the 2,0 or 0,2 analogs. Thus, the sesquiterpene isomer, the number of galactose units, and the galactose arrangement pattern determine the effectiveness of the compound in induction of electrolyte loss and in prevention of toxininduced loss from sugarcane tissues.     

Preparative Electrophoresis of Isotopically Labeled L-Cell Interferons

The preparative method of polyacrylamide gel electrophoresis was adapted for purification and characterization of isotopically labeled L-cell interferons. Re-covery of interferon activity was quantitative, and purification and resolution were comparable to those obtained by analytical polyacrylamide gel electrophoresis. Ultimate specific activities attainable ranged from 2 x 10(6) to 3 x 10(6) international units per mg of protein.     

Purification of rat pancreatic lipase

A procedure for the isolation of lipase (glycerolester hydrolase, EC 3.1.1.3) from rat pancreas is described. The purification scheme includes homogenization of the pancreas, centrifugation at 3,000 rpm, centrifugation at 40,000 rpm, DEAE-cellulose chromatography, precipitation of amylase as the amylase-glycogen complex, gel filtration of the amylase-free proteins on Sephadex G-100, and chromatography on carboxymethyl-Sephadex C-50. The enzyme showed only one band on polyacrylamide gel electrophoresis and had a specific activity of 5330 +/- 80 units/mg of protein.     

Modification in the purification of the sex steroid-binding protein of human serum by affinity chromatography

A purification procedure for the sex steroid-binding protein of human serum is described. The procedure is significantly superior to that recently published (K. E. Mickelson, D. C. Teller, and P. H. Pétra, 1978, Biochemistry17, 1409–1415) and should replace it for the routine preparation of homogeneous protein in relatively larger quantities. The steps involved diethylaminoethyl-cellulose chromatography, affinity chromatography on 5α-dihydrotestosterone-17α-hexanyldiaminoethyl-(1,4-butanediol diglycidyl ether)-agarose, and preparative polyacrylamide gel electrophoresis. The most important difference between this new procedure and that previously published is the affinity adsorbent with contains the steroid covalently linked at the 17α-position rather than the 17β-position. This modification allows the purification of at least 12 mg of homogeneous protein per preparation with a 63% total yield. The properties of the homogeneous protein are the same as previously described.     

Purification of α-galactosidase from coconut

α-Galactosidase from coconut endosperm was purified to homogeneity with a 490-fold increase in specific activity. The yield was 70%, and the specific activity was 24.5 units/mg protein. The purification procedure included extraction, acidification, ammonium sulphate fractionation and hydrophobic chromatography. The hydrophobic gel (Sepharose-4B-capranilide) had a capacity of 0.63 mg of α-galactosidase per ml of gel. Purified α-galactosidase was a glycoprotein with a carbohydrate content of 12%. The molar extinction coefficient was 8.7 x 10⁴/M/cm.     

Comparison of methods for the production and purification of toxin A from Clostridium difficile

Abstract The production and purification of toxin A from Clostridium difficile were studied. When the toxin was produced in dialysis culture it preicipitated quantitatively at pH 5.5 and after purification it appeard homogeneous in polyacrylamide gel electrophoresis (PAGE). The toxin probably consists of two noncovalently bound peptides, each with a molecular mass of about 250 dDa. It is resistant to trypsin but sensitive to papain and chymotrypsin. In contrast, toxin A produced in anaerbic chamber culture precipitated poorly at pH 5.5 (yield 14%) and easily formed aggregates as observed in gel filtration and PAGE Accordingly, dialysis culture seems to be a better method for producing and purifying toxin A.     

Ring formation of perfringolysin o as revealed by negative stain electron microscopy

Perfringolysin O revealed ring- and arc-shaped structures in the absence of cholesterol by negative staining electron microscopy, while before activation with cysteine it showed indistinct arcs and irregularly curved sticks but no rings. These structures were observed only at high concentrations (more than 17 000 hemolytic units per ml) and seemed to be particle associates with 20–28 particles (about 4 nm per particle) linked in a circle. The toxin produced an inactive and high molecular weight complex in the presence of phosphotungstic acid, which was isolated by Sephadex gel filtration. These findings suggest that the rings are the toxin-phosphotungstic acid complexes produced during specimen preparation on a grid in vacuo. The toxin lost the properties necessary for ring formation though moderate modification with glutaraldehyde, showing spindle- and egg-shaped particles of about 4 nm in minor and 5 nm in major axis by negative staining. These facts suggest that the aldehyde modifies the binding sites for phosphotungstic acid, which probably are the basic groups of the toxin molecules. In the presence of cholesterol, even at a low concentration, the toxin revealed rings and arcs by negative staining and also by carbon shadowing electron microscopy, although the toxin itself did not show any characteristic structure without phosphotungstic acid. These observations suggest that the rings are the toxin-cholesterol complexes themselves. The toxin-phosphotungstic acid complexes seemed to have a structure of a single layer of particle associates, while that of the toxin-cholesterol complexes may consist of double or triple layers of the associates because its border was thicker and more distinct.