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Cation-exchange Chromatography (cation-exchange + chromatography)
Selected AbstractsWhey Protein Isolate and ,-Lactalbumin Recovery from Lactic Acid Whey Using Cation-Exchange ChromatographyJOURNAL OF FOOD SCIENCE, Issue 2 2004K. N. Turhan ABSTRACT: The objective of this work was to develop a process to fractionate proteins from lactic acid whey, which is underused compared with sweet whey, using food-grade buffers and cation-exchange chromatography. Bound proteins were desorbed either all at once to make whey protein isolate (WPI), or in 2 steps to 1st make ,-lactalbumin (ALA) and then WPI depleted in ALA. Recovery and purity using lactic acid whey were comparable to previously reported processes using sweet whey. Capacity and throughput were slightly lower using lactic acid whey. This research provides a basis for adding value to the 6 million metric tons of lactic acid whey produced annually in the United States. [source] Growth of Enterococcus mundtii ST15 in medium filtrate and purification of bacteriocin ST15 by cation-exchange chromatographyJOURNAL OF BASIC MICROBIOLOGY, Issue 6 2005Monique Granger Bacteriocin ST15 (bacST15), produced by Enterococcus mundtii ST15, inhibited the growth of a variety of bacteria, including exopolysaccharide (EPS)-producing strains isolated from biofilms in stainless steel pipes. Maximal production of bacST15 (51200 AU/ml) was recorded after 20 h of growth in MRS broth (Biolab), which was maintained throughout fermentation. Only 12800 AU/ml bacST15 has been recorded in MRS filtrate with components smaller than 8000 Da, suggesting that nutrients larger than 8000 Da are required for optimal bacST15 production. Cation-exchange chromatography yielded an active peptide, which is 3944.00 Da, according to electron-spray mass spectrometry and tricin-SDS PAGE. BacST15 is smaller than the 4287 Da reported for bacteriocins ATO6 and KS produced by E. mundtii . The iso-electric point of bacST15 is between 7 and 9, and similar to that reported for pediocin PD-1. (© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Separation of proteins with a molecular mass difference of 2,kDa utilizing preparative double-inverted gradient polyacrylamide gel electrophoresis under nonreducing conditions: Application to the isolation of 24,kDa human growth hormoneELECTROPHORESIS, Issue 23 2005Juan J. Bustamante Abstract A method for separating proteins with a molecular mass difference of 2,kDa using SDS-PAGE under nonreducing conditions is presented. A sample mixture containing several human growth hormone (hGH) isoforms was initially separated on a weak anion-exchange column. Fractions rich in 24,kDa hGH as determined by analytical SDS-PAGE were pooled and further separated by cation-exchange chromatography. The fractions pooled from the cation-exchange chromatography contained two hGH isoforms with a 2,kDa molecular mass difference according to SDS-PAGE analysis, 22 and 24,kDa hGH. The 22 and 24,kDa hGH were separated using continuous-elution preparative double-inverted gradient PAGE (PDG-PAGE) under nonreducing conditions. The preparative electrophoresis gel was composed of three stacked tubular polyacrylamide matrices, a 4% stacking gel, a 13,18% linear gradient gel, and a 15,10% linear inverted gradient gel. Fractions containing purified 24,kDa hGH were pooled and Western blot analysis displayed immunoreactivity to antihGH antibodies. PDG-PAGE provides researchers with an electrophoretic technique to preparatively purify proteins under nonreducing conditions with molecular mass differences of 2,kDa. [source] Efficiency of acid phosphatases secreted from the ectomycorrhizal fungus Hebeloma cylindrosporum to hydrolyse organic phosphorus in podzolsFEMS MICROBIOLOGY ECOLOGY, Issue 2 2010Julien Louche Abstract Ectomycorrhizal fungi may improve the phosphate nutrition of their host plants by secreting, into the soil solution, acid phosphatases (AcPases) able to release orthophosphate (Pi) from soil organic phosphorus (Po). Using cation-exchange chromatography, we separated four fractions with AcPase activity secreted by the ectomycorrhizal fungus Hebeloma cylindrosporum grown in a pure culture under P-starved conditions. Each AcPase active fraction displayed strong ability in vitro to hydrolyse a wide range of phosphate monoesters, but none of them efficiently hydrolysed phytate. Their efficiency to release Pi from soil NaHCO3 -extractable Po was studied in a sandy podzol used intact or autoclaved. Soils were collected in a 15-year-old Pinus pinaster stand, receiving regular fertilization or not. Autoclaving increased the NaHCO3 -extractable Po concentrations by 55% in unfertilized and by 32,43% in fertilized soils. The efficiency of each AcPase fraction was affected significantly by the soil fertilization regime and the soil treatment (intact vs. autoclaved). The proportion of labile Po enzyme ranged from 0% to 11% and 14% to 48% after 1 h of incubation in bicarbonate solutions extracted from intact and autoclaved soils, respectively. This work suggests that AcPases secreted from H. cylindrosporum could be efficient in recycling Po pools from soil microorganisms that may be delivered by soil autoclaving. [source] Growth of Enterococcus mundtii ST15 in medium filtrate and purification of bacteriocin ST15 by cation-exchange chromatographyJOURNAL OF BASIC MICROBIOLOGY, Issue 6 2005Monique Granger Bacteriocin ST15 (bacST15), produced by Enterococcus mundtii ST15, inhibited the growth of a variety of bacteria, including exopolysaccharide (EPS)-producing strains isolated from biofilms in stainless steel pipes. Maximal production of bacST15 (51200 AU/ml) was recorded after 20 h of growth in MRS broth (Biolab), which was maintained throughout fermentation. Only 12800 AU/ml bacST15 has been recorded in MRS filtrate with components smaller than 8000 Da, suggesting that nutrients larger than 8000 Da are required for optimal bacST15 production. Cation-exchange chromatography yielded an active peptide, which is 3944.00 Da, according to electron-spray mass spectrometry and tricin-SDS PAGE. BacST15 is smaller than the 4287 Da reported for bacteriocins ATO6 and KS produced by E. mundtii . The iso-electric point of bacST15 is between 7 and 9, and similar to that reported for pediocin PD-1. (© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] PURIFICATION AND CHARACTERIZATION OF ,-CARRAGEENASE FROM MARINE BACTERIUM MUTANT STRAIN PSEUDOALTEROMONAS SP.JOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2010AJ5-13 AND ITS DEGRADED PRODUCTS ABSTRACT A ,-carrageenan-degrading bacterial strain AJ5 isolated from the intestine of Apostichopus japonicus was identified as Pseudoalteromonas sp. based on the phenotypic characters and 16S rRNA gene sequencing. The mutant Pseudoalteromonas sp. AJ5-13 with ,-carrageenase activity of 61 U/mg protein was obtained from Pseudoalteromonas sp. AJ5 using mutagenesis technique. An extracellular ,-carrageenase was purified from Pseudoalteromonas sp. AJ5-13 cultural supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadex G-200) and cation-exchange chromatography (CM-cellulose 52). The purified enzyme yielded a single band on SDS-PAGE with the molecular mass of 35 kDa. Data of the N-terminal amino acid sequence indicated that this protein might be a novel ,-carrageenase. The pI and Km of the enzyme were 8.5 and 9.8 ± 0.2 mg/mL, respectively. The enzyme exhibited maximal activity at pH 8.0 and 55C. It hydrolyzed the ,-1, 4-glycosidic linkages of ,-carrageenan yielding ,-neocarrabiose, -tetraose, -hexaose, -octaose and -decaose sulfates as the main end-products. PRACTICAL APPLICATIONS ,-Carrageenases degrade ,-carrageenan by hydrolyzing the ,-1,4 linkages to a series of oligosaccharides. Thus, it is expected that like other ,-carrageenases, the ,-carrageenase isolated from Pseudoalteromonas sp. AJ5-13 would also be useful in seaweed biotechnology, pharmacy and immunology. ,-Carrageenases can be applied to study the composition and structure of carrageenans from different red alga, and to study the bacterial ,-carrageenan metabolism. They also provide the opportunity to investigate the structure-function relationship of the hydrolases that degrade self-associating sulfated polysaccharides. Examples of the practical applications of ,-carrageenases include their use in degrading the cell walls of seaweeds to obtain protoplasts, and in hydrolyzing ,-carrageenan to produce oligosaccharides. ,-Carrageenan-oligosaccharides have various potential biological properties, such as antiviral, antitumor, antioxidant activities, cytoprotection, immunomodulation, etc. [source] Whey Protein Isolate and ,-Lactalbumin Recovery from Lactic Acid Whey Using Cation-Exchange ChromatographyJOURNAL OF FOOD SCIENCE, Issue 2 2004K. N. Turhan ABSTRACT: The objective of this work was to develop a process to fractionate proteins from lactic acid whey, which is underused compared with sweet whey, using food-grade buffers and cation-exchange chromatography. Bound proteins were desorbed either all at once to make whey protein isolate (WPI), or in 2 steps to 1st make ,-lactalbumin (ALA) and then WPI depleted in ALA. Recovery and purity using lactic acid whey were comparable to previously reported processes using sweet whey. Capacity and throughput were slightly lower using lactic acid whey. This research provides a basis for adding value to the 6 million metric tons of lactic acid whey produced annually in the United States. [source] Selective isolation of multiple positively charged peptides for 2-DE-free quantitative proteomicsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2006Aniel Sánchez Abstract A method for quantitative proteomic analysis based on the selective isolation of multiply charged peptides (RH peptides) containing arginine and histidine residues is described. Two pools of proteins are digested in tandem with lysyl-endopeptidase and trypsin and the primary amino groups of proteolytic peptides are separately labeled with d3 - and d0 -acetic anhydride. This reaction has a dual purpose: (i) to allow the relative protein quantification in two different conditions and (ii) to restrict the positive charges of peptides to the presence of arginine and histidine. The N -acylated peptides are separated by cation-exchange chromatography into two groups, neutral and singly charged peptides (R,+,H,,,1) that are neither retained nor analyzed, whereas the multiply charged peptides (R,+,H>1) are retained into the column and can be eluted in batch or further fractionated using a saline gradient before LC-MS/MS analysis. In silico analysis revealed that the selective isolation of RH peptides considerably simplifies the complex mixture of peptides (three RH peptides/protein) and at the same time they represent 84% of the whole proteomes. The selectivity, and recovery of the method were evaluated with model proteins and with a complex mixture of proteins extracted from Vibrio cholerae. [source] Comparison of alternative analytical techniques for the characterisation of the human serum proteome in HUPO Plasma Proteome ProjectPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2005Xiaohai Li Abstract Based on the same HUPO reference specimen (C1-serum) with the six proteins of highest abundance depleted by immunoaffinity chromatography, we have compared five proteomics approaches, which were (1) intact protein fractionation by anion-exchange chromatography followed by 2-DE-MALDI-TOF-MS/MS for protein identification (2-DE strategy); (2) intact protein fractionation by 2-D HPLC followed by tryptic digestion of each fraction and microcapillary RP-HPLC/microESI-MS/MS identification (protein 2-D HPLC fractionation strategy); (3) protein digestion followed by automated online microcapillary 2-D HPLC (strong cation-exchange chromatography (SCX)-RPC) with IT microESI-MS/MS; (online shotgun strategy); (4) same as (3) with the SCX step performed offline (offline shotgun strategy) and (5) same as (4) with the SCX fractions reanalysed by optimised nanoRP-HPLC-nanoESI-MS/MS (offline shotgun-nanospray strategy). All five approaches yielded complementary sets of protein identifications. The total number of unique proteins identified by each of these five approaches was (1) 78, (2) 179, (3) 131, (4) 224 and (5) 330 respectively. In all, 560 unique proteins were identified. One hundred and sixty-five proteins were identified through two or more peptides, which could be considered a high-confidence identification. Only 37 proteins were identified by all five approaches. The 2-DE approach yielded more information on the pI -altered isoforms of some serum proteins and the relative abundance of identified proteins. The protein prefractionation strategy slightly improved the capacity to detect proteins of lower abundance. Optimising the separation at the peptide level and improving the detection sensitivity of ESI-MS/MS were more effective than fractionation of intact proteins in increasing the total number of proteins identified. Overall, electrophoresis and chromatography, coupled respectively with MALDI-TOF/TOF-MS and ESI-MS/MS, identified complementary sets of serum proteins. [source] Structure of the mexicain,E-64 complex and comparison with other cysteine proteases of the papain familyACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2007J. A. Gavira Mexicain is a 23.8,kDa cysteine protease from the tropical plant Jacaratia mexicana. It is isolated as the most abundant product after cation-exchange chromatography of the mix of proteases extracted from the latex of the fruit. The purified enzyme inhibited with E-64 [N -(3-carboxyoxirane-2-carbonyl)-leucyl-amino(4-guanido)butane] was crystallized by sitting-drop vapour diffusion and the structure was solved by molecular replacement at 2.1,Ĺ resolution and refined to an R factor of 17.7% (Rfree = 23.8%). The enzyme belongs to the ,+, class of proteins and the structure shows the typical papain-like fold composed of two domains, the ,-helix-rich (L) domain and the ,-barrel-like (R) domain, separated by a groove containing the active site formed by residues Cys25 and His159, one from each domain. The four monomers in the asymmetric unit show one E-64 molecule covalently bound to Cys25 in the active site and differences have been found in the placement of E-64 in each monomer. [source] Incorporation of methyl-protonated valine and leucine residues into deuterated ocean pout type III antifreeze protein: expression, crystallization and preliminary neutron diffraction studiesACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010Isabelle Petit-Haertlein Antifreeze proteins (AFPs) are found in different species from polar, alpine and subarctic regions, where they serve to inhibit ice-crystal growth by adsorption to ice surfaces. Recombinant North Atlantic ocean pout (Macrozoarces americanus) AFP has been used as a model protein to develop protocols for amino-acid-specific hydrogen reverse-labelling of methyl groups in leucine and valine residues using Escherichia coli high-density cell cultures supplemented with the amino-acid precursor ,-ketoisovalerate. Here, the successful methyl protonation (methyl reverse-labelling) of leucine and valine residues in AFP is reported. Methyl-protonated AFP was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Crystals were grown in D2O buffer by the sitting-drop method. Preliminary neutron Laue diffraction at 293,K using LADI-III at ILL showed in a few 24,h exposures a very low background and clear small spots up to a resolution of 1.80,Ĺ from a crystal of dimensions 1.60 × 0.38 × 0.38,mm corresponding to a volume of 0.23,mm3. [source] Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze proteinACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009Isabelle Petit-Haertlein The highly homologous type III antifreeze protein (AFP) subfamily share the capability to inhibit ice growth at subzero temperatures. Extensive studies by X-ray crystallography have been conducted, mostly on AFPs from polar fishes. Although interactions between a defined flat ice-binding surface and a particular lattice plane of an ice crystal have now been identified, the fine structural features underlying the antifreeze mechanism still remain unclear owing to the intrinsic difficulty in identifying H atoms using X-ray diffraction data alone. Here, successful perdeuteration (i.e. complete deuteration) for neutron crystallographic studies of the North Atlantic ocean pout (Macrozoarces americanus) AFP in Escherichia coli high-density cell cultures is reported. The perdeuterated protein (AFP D) was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Well shaped perdeuterated AFP D crystals have been grown in D2O by the sitting-drop method. Preliminary neutron Laue diffraction at 293,K using LADI-III at ILL showed that with a few exposures of 24,h a very low background and clear small spots up to a resolution of 1.85,Ĺ were obtained using a `radically small' perdeuterated AFP D crystal of dimensions 0.70 × 0.55 × 0.35,mm, corresponding to a volume of 0.13,mm3. [source] Dynamics of protein uptake within the adsorbent particle during packed bed chromatographyBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2002Jürgen Hubbuch Abstract A new experimental set-up for on-line visualization of the intra-particle uptake kinetics during packed bed chromatography has been designed and tested. Confocal laser scanning microscopy was used to analyze the dynamics of protein adsorption to porous stationary phases. In combination with this, a flow cell was developed that could be packed with chromatography media and operated as a fully functional mini-scale chromatography column. Adsorption profiles of single- and two-component mixtures containing BSA and IgG 2a during packed bed cation-exchange chromatography were investigated. The two proteins appear to exhibit different transport characteristics. For BSA a classical "shrinking core" behavior could be detected. The profiles obtained during IgG 2a adsorption point toward a different transport mode, which deviates from the classical pore-diffusion picture. For the two-component system, a superposition of the single-component profiles combined with a classical displacement of the weaker bound protein species was found. The results indicate that depending on the adsorbed protein the uptake can vary tremendously, even for adsorption to the same chromatographic support. It is clearly shown that the new microcolumn allows in situ quantitative investigations of protein adsorption dynamics within a single particle, which adds a new tool to the available methods for characterizing and optimizing protein adsorption chromatography. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 359,368, 2002. [source] Development of a high throughput protein a well-plate purification method for monoclonal antibodiesBIOTECHNOLOGY PROGRESS, Issue 5 2009Jennifer Hopp Abstract We have developed a new high throughput method for the purification of monoclonal antibodies from harvested cell culture fluid for analytical characterization. This method uses Protein A resin in a 96 well-plate format with protein loading sufficient to perform multiple analyses per well. Resin and buffer conditions were optimized to obtain aggregate and charge variant comparability with three preparative Protein A purified monoclonal antibodies. We are able to successfully demonstrate comparability for aggregate within 0.25% based upon size-exclusion chromatography. Acidic species were found to be within 2% from the preparative purified control based upon cation-exchange chromatography, 5% based upon capillary zone electrophoresis, and 3% based upon imaged capillary isoelectric focusing. Glycan distribution was analyzed and was within 1% of the preparative purified controls. A tryptic digest was performed and all peaks in the preparative purified control were found in the first elution from the well-plate format. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] What is the role of the hevein-like domain of fruit class I chitinases in their allergenic capacity?CLINICAL & EXPERIMENTAL ALLERGY, Issue 3 2002A. Děaz-Perales Background Class I chitinases are the major panallergens in fruits associated with the latex,fruit syndrome. These enzymes contain an N-terminal hevein-like domain homologous to latex hevein, and a larger catalytic domain. The role of these domains in their allergenic capacity is still controversial. Objective We sought to evaluate the role of both domains of class I chitinases in their IgE-binding properties, using Cas s 5, the major allergen from chestnut, as a model. Methods Recombinant Cas s 5 and its deleted form, lacking the hevein-like domain, designated rCat, were expressed in Pichia pastoris using the pPIC 9 vector. Both recombinant products were purified from the supernatants of transformed yeast cultures by gel-filtration and cation-exchange chromatography. The isolated proteins were characterized by N-terminal sequencing, enzymatic activity and N-glycosylation tests, anti-chitinase and specific IgE immunodetection. Immunoblot, RAST and CAP inhibition assays were also performed. Results Both purified rCas s 5 and rCat showed the expected N-terminal amino acid sequences and an enzymatic activity similar to that of their natural counterparts isolated from chestnut seeds, and were strongly recognized by anti-chitinase antibodies. In contrast, only rCas s 5, but not rCat, bound specific IgE from sera of patients suffering from the latex,fruit syndrome, and fully inhibited IgE-binding to natural Cas s 5 in immunoblot inhibition assays. Latex hevein also exerted a strong immunoblot inhibition of IgE-binding to chestnut Cas s 5. RAST and CAP inhibition using whole chestnut extract on the solid phase, rendered inhibition levels around 70,90% for rCas s 5 and 60% for rCat, in contrast to the immunoblotting results. Conclusions Recombinant Cas s 5 behaves like natural Cas s 5 in IgE-binding assays in vitro. The hevein-like domain of allergenic class I chitinases seems to include all their main IgE-binding epitopes when tested by immunodetection and immunoblot inhibition experiments. RAST and CAP inhibition assays, on the contrary, suggest that relevant epitopes are also harboured in the catalytic domain of these allergens. [source] | ||||||||||||||||||||||