Identification of a small antimycotic peptide produced by Bacillus amyloliquefaciens 6256.

Bacillus sp. 6256 is a good biocontrol agent against Botrytis cinerea which caused tomato gray mold disease. Strain 6256 was identified as B. amyloliquefaciens by analysis of its partial gyrB gene sequence. To identify and characterize the antimycotic peptides from the culture broth of the bacterium, the antimicrobial substances produced by B. amyloliquefaciens 6256 were isolated by ammonium sulfate precipitation, Superdex 200 gel filtration chromatography and DEAE anion exchange chromatography. The purified compound was designated as P657. The biological activity of P657 was stable at as high as 100 °C for 20 min and in pH value ranged from 5 to 10. The antimycotic compound was resistant to trypsin and proteinase K, and could completely inhibit spore germination of Botrytis cinerea in vitro. MALDI-TOF-MS analysis results showed the presence of fengycins A (C16-C17) and fengycins B (C15-C17) isoforms in P657.

Partial purification and characterization of a novel histidine decarboxylase from Enterobacter aerogenes DL-1.

Histidine decarboxylase (HDC) from Enterobacter aerogenes DL-1 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 52.4 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for HDC activity was 6.5, and the enzyme was stable between pH 4 and 8. Enterobacter aerogenes HDC had optimal activity at 40°C and retained most of its activity between 4 and 50°C. HDC activity was reduced in the presence of numerous tested compounds. Particularly with SDS, it significantly (p < 0.01) inhibited enzyme activity. Conversely, Ca(2+) and Mn(2+) showed prominent activation effects (p < 0.01) with activity increasing to 117.20% and 123.42%, respectively. The Lineweaver-Burk plot showed that K m and V max values of the enzyme for L-histidine were 0.21 mM and 71.39 µmol/min, respectively. In comparison with most HDCs from other microorganisms and animals, HDC from E. aerogenes DL-1 displayed higher affinity and greater reaction velocity toward L-histidine.

New method for the speciation of ruthenium-based chemotherapeutics in human serum by conjoint liquid chromatography on affinity and anion-exchange monolithic disks.

An important step in pharmacological characterisation of a candidate drug is the study of the drugs interactions with serum proteins. In the present work, conjoint liquid chromatography (CLC) was used for separation of ruthenium (Ru)-based drug candidates in human serum. CIM Protein G and CIM DEAE disks were assembled together in a single housing forming a CLC monolithic column. By applying isocratic elution with Tris-HCl-NaHCO3 buffer (pH 7.4) in the first min, followed by gradient elution with 1 mol L(-1) NH4Cl (pH 7.4) in the next 9 min, immunoglobulins (IgG) were retained by the Protein G disk enabling subsequent separation of unbound Ru species from Ru species bound to human serum transferrin (Tf) and albumin (HSA) on the CIM DEAE disk. Finally, elution with acetic acid (AcOH) in the next 3 min allowed separation of Ru species associated with IgG. Protein elution was followed on-line with UV detection at 278nm, while the separated Ru species were quantified by post-column isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS). The instrumental set-up enabled fast two-dimensional separation by affinity and ion-exchange modes to be carried out in a single chromatographic run. Two Ru-based chemotherapeutics: a newly synthesised compound chlorido(η6-p-cymene)(nalidixicato-κ2O,O)Ru(II) (1) and (H2im)[trans-Ru(III)Cl4(Him)2] (2; KP418), which is currently undergoing preclinical studies, were investigated. The CLC procedure applied is sensitive with low limit of detection (LOD) (0.027 µg Ru mL(-1) for (1)) and good method repeatability (RSD±3.5%). The experimental data revealed that it enables investigation of the kinetics of interaction of positively charged and neutral complexes of metallodrugs with serum proteins as well as the distribution of metallodrug species in human serum. However, negatively charged metallic complexes co-eluted with Tf and HSA and thus hindered their speciation analysis. An example of successful application of the kinetic studies on the CLC column is (1), a neutral Ru complex that hydrolyses to a positively charged species. For comparison, speciation data obtained for serum samples spiked with cisplatin are also shown.

High capacity cryogel-type adsorbents for protein purification.

Cryogel bodies were modified to obtain epoxy groups by graft-copolymerization using both chemical and gamma irradiation initiation techniques. The free epoxy adsorbents were reacted further to introduce diethylaminoethanol (DEAE) functionalities. The resulting weak anion-exchange cryogel adsorbents showed dynamic binding capacities of ca. 27±3mg/mL, which was significantly higher than previously reported for this type of adsorbent material. Gamma irradiated grafting initiation showed a 4-fold higher capacity for proteins than chemical grafting initiation procedures. The phosphate capacity for these DEAE cryogels was 119mmol/L and also showed similar column efficiency as compared to commercial adsorbents. The large pores in the cryogel structure ensure convective transport of the molecules to active binding sites located on the polymer-grafted surface of cryogels. However, as cryogels have relatively large pores (10-100µm), the BET area available for surface activation is low, and consequently, the capacity of the cryogels is relatively low for biomolecules, especially when compared to commercial beaded adsorbents. Nevertheless, we have shown that gamma ray mediated surface grafting of cryogel matrices greatly enhance their functional and adsorptive properties.

[Extraction and purification of acidic polysaccharide from Moerella iridescens].

OBJECTIVE: To optimize extraction and purification methods of acidic polysaccharide from Moerella iridescens (MIAP). METHODS: With alkali extraction process and orthogonal experiment,the time consumption,temperature,pH value of the solution and alcohol concentration during the extraction were optimized. The crude products were deprived of protein,pigment and ion,then were purified with DEAE-cellulose ion-exchange chromatography and verified with Sephadex G-100 and cellulose acetate membrane electrophoresis,and examined with infrared spectrum. RESULTS: The optimized extraction conditions were as follows: extraction time 6 h,extraction temperature 70 degree,the solution pH 8.0 and the concentration of alcohol precipitation 70%. Intuitive features showed that the MIAP was pure white crystalline granular with slight dark brown color. The purification results demonstrated that the target MIAP was eluted and identified as a homogeneous components by DEAE-cellulose ion exchange column,Sephadex G-100 and cellulose acetate membrane electrophoresis. Infrared spectral scanning suggested that MIAP was α-D-type terminated glucopyranose. Intuitive features showed that MIAP was soft and cottony white. CONCLUSION: The extraction process with orthogonal test has been optimized and the acidic polysaccharide from Moerella iridescens is successfully isolated.

Purification and characterization of a novel laccase from the mushroom Pleurotus nebrodensis.

A novel laccase with a molecular mass of 64 kDa and the N-terminal sequence AIGPDDTINF was isolated from fresh fruiting bodies of the mushroom Pleurotus nebrodensis. The purification protocol comprised ion exchange chromatography on DEAE-cellulose, CM-cellulose, and Q-Sepharose, and gel filtration on Superdex 75. The laccase was adsorbed on DEAE-cellulose and Q-Sepharose, but not on CM-cellulose. It demonstrated an optimal temperature of 70°C. The enzyme activity increased steadily over the temperature range 20°C-70°C. There was only a slight reduction in activity at 80°C. However, all activity disappeared following exposure to 100°C for 10 minutes. The enzyme activity changed only slightly over the pH range 3-5, with the optimum at pH 5, but underwent a precipitous decline when the pH was elevated to 6, and was undetectable at pH 8 and pH 9.

Effects of ligand density and pore size on the adsorption of bovine IgG with DEAE ion-exchange resins.

Immunoglobulin G is an important plasma protein with many applications in therapeutics and diagnostics, which can be purified effectively by ion exchange chromatography. The ligand densities and pore properties of ion-exchange resins have significant effects on the separation behaviors of protein, however, the understandings are quite limited. In this work, with bovine immunoglobulin as the model IgG, the adsorption isotherms and adsorption kinetics were investigated systematically with series of diethylaminoethyl ion-exchange resins with different ligand densities and pore sizes. The Langmuir equation and pore diffusion model were used to fit the experimental data. The influences of ligand density and pore size on the saturated adsorption capacity, the dissociation constant and the effective diffusivity were discussed. The adsorption capacities increased with the increase of ligand density and the decrease of pore size, and an integrative parameter was proposed to describe the combined effects of ligand density and pore size. It was also found that the effective pore diffusion coefficient of the adsorption kinetics was influenced by pore sizes of resins, but was relatively independent on the ligand densities of resins. For a given protein, the ligand density and pore size should be optimized for improving the protein adsorption.

Purification and characterization of phytase with a wide pH adaptation from common edible mushroom Volvariella volvacea (Straw mushroom).

A novel phytase with a molecular mass of 14 kDa was isolated from fresh fruiting bodies of the common edible mushroom Volvariella volvacea (Straw mushroom). The isolation procedure involved successive chromatography on DEAE-cellulose, CM-cellulose, Affi-gel blue gel, Q-Sepharose and Superdex-75. The enzyme was a monomeric protein and was unadsorbed on DEAE-cellulose, CM-cellulose and Affi-gel blue gel, but was adsorbed on Q-Sepharose. The enzyme was purified 51.6-fold from the crude extract with 25.9% yield. Its N-terminal amino acid sequence GEDNEHDTQA exhibited low homology to the other reported phytases. The optimal pH and temperature of the purified enzyme was 5 and 45 degrees C, respectively. The enzyme was quite stable over the pH range of 3.0 to 9.0 with less than 30% change in its activity, suggesting that it can be used in a very wide pH range. The enzyme exhibited broad substrate selectivity towards various phosphorylated compounds, but lacked antifungal activity against tested plant pathogens.

Development of a process for large-scale purification of C-phycocyanin from Synechocystis aquatilis using expanded bed adsorption chromatography.

In this paper a large and scaleable method for purification of C-phycocyanin (C-PC) from the cyanobacteria Synechocystis aquatilis has been developed. Phycobiliproteins are extracted from the cells by osmotic shock and separated by passing the centrifuged cell suspension through an expanded bed adsorption chromatography (EBAC) column using Streamline-DEAE as adsorbent. The eluted C-PC rich solution is finally purified by packed-bed chromatography using DEAE-cellulose. Optimal extraction is achieved using phosphate 0.05 M buffer pH 7.0 twice. The operation of EBAC is optimized on a small scale using a column of 15 mm internal diameter (I.D.). The optimal conditions are a sample load of 4.9 mg C-PC/mL adsorbent, an expanded bed volume twice the settled bed volume and a sample viscosity of 1.020 mP. The EBAC process is then scaled up by increasing the column I.D. (15, 25, 40, 60 and 90 mm) and the success of the scale-up process is verified by determining the protein breakthrough capacity and product recovery. The yield of the EBAC step is in the range of 90-93% for every column diameter. To obtain pure C-PC, conventional ion-exchange chromatography with DEAE-cellulose is utilized and a yield of 74% is obtained. The overall yield of the process, comprising all steps, is 69%. The purification steps are monitored using SDS-PAGE and the purity of recovered C-PC is confirmed by absorption and emission spectroscopy and RP-HPLC. Results show that EBAC method is a scalable technology that allows large quantities of C-PC to be obtained without product loss, maintaining a high protein recovery while reducing both processing cost and time.

Large-scale production, purification and bioactivity assay of recombinant human interleukin-6 in the methylotrophic yeast Pichia pastoris.

A DNA fragment containing the mature human interleukin (IL)-6 sequence was cloned into pPICZαA, generating a fusion protein with the alpha factor from baker's yeast and integrated into the genome of Pichia pastoris strain X-33. Recombinant yeast transformants with high-level rhIL-6 production were identified, secreting as much as 280 mg L(-1) rhIL-6 after 4 days of induction by methanol. The rhIL-6 was purified by PEG-8000 precipitation, followed by DEAE anion exchange and Sephadex G-75 gel filtration, yielding over 95% pure rhIL-6 at about 170 mg L(-1) . Mass spectrometry analysis showed that the rhIL-6 has a molecular weight of 20,908.85 Da, which is close to the mass calculated from the sequence of the protein. Functional analysis of the purified rhIL-6 using the lymphocyte proliferation assay by an MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl-tetrazoliumbromide] method demonstrated a specific activity that is at least fivefold higher than the commercial rhIL-6 produced in Escherichia coli. In summary, the experimental procedure we have reported here allows us to obtain a large amount of rhIL-6 from P. pastoris suitable for subsequent biophysical studies.

Adiponectin does not bind to gelatin: a new and easy way to purify high-molecular-weight adiponectin from human plasma.

Human plasma contains three forms of adiponectin, a trimer, a hexamer, and a high-molecular-weight (HMW) multimer. We previously reported HMW adiponectin was a gelatin-binding protein of 28 kDa (GBP28), it having been purified due to its affinity to gelatin-Cellulofine (Nakano, Y., et al. Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma. J. Biochem. 1996. 120: 803-12). Although HMW adiponectin binds to gelatin-Cellulofine, it cannot bind to gelatin-Sepharose. Gelatin-Cellulofine was made of formyl-Cellulofine and gelatin, and we found that HMW adiponectin binds to reduced formyl-Cellulofine with similar affinity as to gelatin-Cellulofine. Through only two steps using reduced formyl-Cellulofine and DEAE-Sepharose, HMW adiponectin can be effectively purified from human plasma.

Antibody purification: ion-exchange chromatography.

Ion exchange chromatography techniques are the focus of this chapter and they showcase the power of this method for the purification of proteins and monoclonal antibodies. The technique is powerful and can separate biomolecules that have minor differences in their net charge, e.g., two protein molecules differing by a single charged amino acid. Given the amphoteric character of proteins the pH of the solution is important in the determination of the type of ion exchanger used. Immunoglobulins, although they can be purified by either cation or anion exchange chromatography, are most frequently purified by anion exchange with DEAE resins. The purification of the rabbit IgG fraction from serum using a DEAE column is detailed as well as the purification of IgG from ascitic fluid using FPLC, from loading to elution of the purified and concentrated protein.

High-sensitivity express immunochromatographic method for detection of plant infection by tobacco mosaic virus.

A highly sensitive express immunochromatography method for molecular diagnosis of plant virus infections was elaborated on the example of a model object - tobacco mosaic virus (TMV). The analysis time does not exceed 5 min, and the lower limit of TMV detection in non-clarified leaf extract (2-4 ng/ml) is comparable with the sensitivity of the enzyme-linked immunosorbent assay of the virus. A single measurement requires 0.1-0.2 ml tested solution (extract from 10-20 mg of leaf material). The sensitivity of TMV determination in the leaf tissue extract was increased by more than one order of magnitude using signal enhancement by silver and is 0.1 ng/ml. In this case, analysis time did not exceed 25 min. The simplicity of this method makes it especially convenient in express diagnosis of numerous analyzed specimens. The prototype of a diagnostic kit for serial analyses of plant viral infections both in laboratory and field conditions was elaborated.

[Ribonuclease from the hepatopancreas of the red king crab Paralithodes camtschatica].

Three enzyme preparations, two acid and one alkaline RNases, were isolated from the hepatopancreas of the red king crab Paralithodes camtschatica using DEAE-cellulose chromatography and gel chromatography. The alkaline RNase was activated by Mg2+ ions and had a pH optimum of 7.2; the acid RNases, a pH optimum of 5.5. The molecular weight of the alkaline RNase was 19 kDa; two acid RNases, 33 and 70 kDa, respectively. The enzymes exhibited a sufficiently high thermostability (IT50 = 53-55 degrees C) and were strongly inhibited by NaCl (IC50, 0.1-0.25 M). The alkaline RNase exhibited no specificity for heterocyclic bases, whereas the acid RNases hydrolyzed poly(U) and poly(A) at maximum rates.

Purification of restriction fragments containing replication intermediates from complex genomes for 2-D gel analysis.

In order to perform 2-D gel analyses on restriction fragments from higher eukaryotic genomes, it is necessary to remove most of the linear, nonreplicating, fragments from the starting DNA preparation. This is so because the replication intermediates in a single-copy locus constitute such a minute fraction of all of the restriction fragments in a standard DNA preparation - whether isolated from synchronized or asynchronous cultures. Furthermore, the very long DNA strands that characterize higher eukaryotic genomes are inordinately subject to branch migration and shear. We have developed a method that results in significant enrichment of replicating fragments that largely maintain their branched intermediates. The method depends upon two important factors: (1) replicating fragments in higher eukaryotic nuclei appear to be attached to the nuclear matrix in a supercoiled fashion, and (2) partially single-stranded fragments (e.g., those containing replication forks) are selectively adsorbed to benzoylated napthoylated DEAE (BND)-cellulose in high salt conCentrations. By combining matrix-enrichment and BND-cellulose chromatography, it is possible to obtain preparations that are enriched as much as 200-fold over the starting genomic DNA and are thus suitable for analysis on 2-D gels.

Inhibition of the hemorrhagic and proteolytic activities of Lansberg's hognose pit viper (Porthidium lansbergii hutmanni) venom by opossum (Didelphis marsupialis) serum: isolation of Didelphis marsupialis 0.15Dm fraction on DEAE-cellulose chromatography.

Earlier studies have revealed the ability of sera from several mammals to neutralize the toxic effects of snake venom. The Venezuelan opossum (Didelphis marsupialis) is one that has been found to inhibit hemorrhagic and proteolytic activities of venoms from many species of snakes. In this article it is shown that the opossum sera and its 0.15DM fraction were able to completely neutralize both hemorrhagic and hydrolysis (proteolysis) of casein effects induced by venom of the Lansberg's hognose pit viper (Porthidium lansbergii hutmanni). We have used DEAE-cellulose ion exchange chromatography to collect protein fractions from D. marsupialis sera which were able to defend mice from the lethal effects of P.l. hutmanni venom. The fractions separated were homogeneous by conventional electrophoresis using SDS-PAGE. The protein bands obtained contained molecular weights of approximately 6 to 220 kDa. These results revealed the presence of proteases inhibitors in the opossum sera fractions and the inhibition of venom activity by opossum sera suggesting a reciprocal adaptation at the molecular level.

Developing procedures for the large-scale purification of human serum butyrylcholinesterase.

Human serum butyrylcholinesterase (Hu BChE) is the most viable candidate for the prophylactic treatment of organophosphate poisoning. A dose of 200 mg/70 kg is predicted to protect humans against 2x LD(50) of soman. Therefore, the aim of this study was to develop procedures for the purification of gram quantities of this enzyme from outdated human plasma or Cohn Fraction IV-4. The purification of Hu BChE was accomplished by batch adsorption on procainamide-Sepharose-CL-4B affinity gel followed by ion-exchange chromatography on a DEAE-Sepharose column. For the purification of enzyme from Cohn Fraction IV-4, it was resuspended in 25 mM sodium phosphate buffer, pH 8.0, and fat was removed by decantation, prior to batch adsorption on procainamide-Sepharose gel. In both cases, the procainamide gel was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0, containing 0.05 M NaCl, and the enzyme was eluted with the same buffer containing 0.1 M procainamide. The enzyme was dialyzed and the pH was adjusted to 4.0 before loading on the DEAE column equilibrated in sodium acetate buffer, pH 4.0. The column was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0 containing 0.05 M NaCl before elution with a gradient of 0.05-0.2M NaCl in the same buffer. The purity of the enzyme following these steps ranged from 20% to 40%. The purity of the enzyme increased to >90% by chromatography on an analytical procainamide affinity column. Results show that Cohn Fraction IV-4 is a much better source than plasma for the large-scale isolation of purified Hu BChE.

Isolation of new pregnancy-associated glycoproteins from water buffalo (Bubalus bubalis) placenta by Vicia villosa affinity chromatography.

The present study describes the isolation and characterization of new pregnancy-associated glycoprotein molecules (PAG) from midpregnancy and late-pregnancy placentas in the water buffalo (Bubalus bubalis). After extraction, the homogenates are subjected to acid and ammonium sulfate precipitations followed by DEAE chromatography. Subsequently, the water buffalo PAG (wbPAG) from these solutions are enriched by Vicia villosa agarose (VVA) affinity chromatography. As determined by western blotting with anti-PAG sera, the apparent molecular masses of the immunoreactive bands from the VVA peaks range from 59.5 to 75.8kDa and from 57.8 to 73.3kDa in the midpregnancy and late-pregnancy placentas, respectively. Amino-terminal microsequencing of the immunoreactive proteins has allowed the identification of three distinct wbPAG sequences, which have been deposited in the SwissProt database: RGSXLTIHPLRNIRDFFYVG (acc. no. P85048), RGSXLTILPLRNIID (acc. no. P85049), and RGSXLTHLPLRNI (acc. no. P85050). Their comparison to previously identified proteins has shown that two of them are new because they have not been described before. Our results confirm the suitability of VVA chromatography for the enrichment of the multiple PAG molecules expressed in buffalo placenta.

A novel assay system for myeloperoxidase activity in whole saliva.

OBJECTIVES: The application of a novel assay system for the direct measurement of MPO (myeloperoxidase) activity in whole saliva. DESIGN AND METHODS: The assay system employs a novel sensitive substrate from 3,3'-diaminobenzidine (DAB) and guaiacol in the presence of dapsone (4,4'-diaminodiphenylsulfone) to determine MPO activity in whole saliva using an original "sandwich" test-disk (DEAE-cellulose paper and cellulose chromatography paper). The saliva (0.1 mL) was directly applied to the sandwich test-disk, and then 0.1 mL of the substrate solution containing 1 mM dapsone in 0.3 M Tris-HCl buffer (pH 7.5) was added. After incubation for 30 min at room temperature, absorbance on the test-disk was measured at 460 nm with an optical analyzer. RESULTS: The assay system was shown to distinguish MPO from salivary peroxidase in whole mixed saliva and was sensitive, easy and cheap. The assays revealed that MPO activity in whole saliva from subjects with periodontal disease was significantly higher than in saliva from healthy subjects. There was also a significant positive correlation between MPO activity and the probing depth of subgingival pockets (r=0.736, p<0.001). CONCLUSIONS: These results indicate that this novel assay system for measurement of MPO is a useful technique for predicting the progression of periodontal disease.

Direct purification of recombinant hepatitis B core antigen from two different pre-conditioned unclarified Escherichia coli feedstocks via expanded bed adsorption chromatography.

Direct recovery of hepatitis B core antigen (HBcAg) from unclarified Escherichia coli homogenates via expanded bed adsorption chromatography (EBA) has been explored in this study. Streamline DEAE was selected as the anion exchanger to recover HBcAg from heat-treated and non-heat-treated unclarified feedstocks. The use of anion-exchanger for direct extraction of proteins from unclarified feedstock is not preferred due to lack of specificity of its ligand. In this study, thermal treatment of the unclarified feedstock at 60 degrees C has resulted in 1.2- and 1.8-fold increases in yield and purity of HBcAg, respectively, compared with that purified from non-heat-treated feedstock. Heating the crude feedstock has resulted in denaturation and precipitation of contaminants in the feedstock, hence reducing non-specific interactions between the cell debris and adsorbent. The selectivity of the anion-exchanger has also been increased as shown in the breakthrough curve obtained. Enzyme-linked immunosorbent assay showed that the antigenicity of the HBcAg from heat-treated unclarified feedstock is still preserved.