Controlled oxidative protein refolding using an ion-exchange column

Column-based refolding of complex and highly disulfide-bonded proteins simplifies protein renaturation at both preparative and process scale by integrating and automating a number of operations commonly used in dilution refolding. Bovine serum albumin (BSA) was used as a model protein for refolding and oxido-shuffling on an ion-exchange column to give a refolding yield of 55% after 40 h incubation. Successful on-column refolding was conducted at protein concentrations of up to 10 mg/ml and refolded protein, purified from misfolded forms, was eluted directly from the column at a concentration of 3 mg/ml. This technique integrates the dithiothreitol removal, refolding, concentration and purification steps, achieving a high level of process simplification and automation, and a significant saving in reagent costs when scaled. Importantly, the current result suggests that it is possible to controllably refold disulfide-bonded proteins using common and inexpensive matrices, and that it is not always necessary to control protein-surface interactions using affinity tags and expensive chromatographic matrices. Moreover, it is possible to strictly control the oxidative refolding environment once denatured protein is bound to the ion-exchange column, thus allowing precisely controlled oxido-shuffling.     

The quantitative collection and recovery of silica using an ion-exchange column

Summary Results are presented to show the quantitative collection and recovery of microgram quantities of silica, using a strongly basic anion-exchange resin, Amberlite IRA-400. The method is applicable to both metasilicate and fluosilicate solutions, and has been applied to the analysis of sodium alginate solutions for silica content.

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Zusammenfassung Es wird über die Verwendbarkeit des stark basischen Anionenaustauschers Amberlite IRA-400 zur quantitativen Abtrennung und Bestimmung von Mikrogramm-Mengen Siliciumdioxyd berichtet. Das Verfahren eignet sich für Lösungen von Metasilikaten und Silicofluoriden und wurde zur Bestimmung des Siliciumdioxydgehaltes von Natriumalginatlösungen verwendet.

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Résumé On fournit les résultats montrant la récupération quantitative de mioroquantités de silice, utilisant une résine fortement basique pour échange d'anions IRA-400. La méthode est applicable aussi bien aux solutions de métasilicate que de fluosilicates et elle a été utilisée pour le dosage de la silice dans les solutions d'alginate de sodium.

     

Separation of uranium from uraniferous coaly clays using a selective ion-exchange two-step elution system

Summary An ion-exchange process for the selective separation and enrichment of uranium from the main elements Si, Al, Fe, Ca, Mg, Na, K as well as from Mo and V, which are present in uraniferous coaly clays, has been developed. After a selective carbonate leaching of the roasted ore, a 0.5 mol/l Na₂CO₃/0.5 mol/l NaHCO₃ solution was passed through the macroporous ion-exchanger AG MP-1, which at pH10 absorbed uranium quantitatively in form of a carbonato complex. Remaining absorbed amounts of Mo and V were eluted with 0.1 mol/l EDTA in 0.5 mol/l Na₂CO₃/0.5 mol/l NaHCO₃, while U was quantitatively separated by a second elution step with 0.5 mol/l HNO₃ and was afterwards precipitated with NH₄OH as a high-grade yellow cake. Differential pulse polarography (DPP), atomic absorption spectroscopy (AAS), X-ray fluorescence (XRF), instrumental neutron activation analysis (INAA) and inductively coupled plasma mass spectrometry (ICP-MS) had been used to determine the uranium content in the raw materials and to investigate the effectivity of the different steps of the developed process.     

Purification and characterization of a nuclease from Lentinus edodes.

An endonuclease with 3'-nucleotidase activity (nuclease Le1) was purified from fruit bodies of Lentinus edodes in a single band on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The apparent molecular weight of nuclease Le1 was about 27000. The nuclease was inactivated in the presence of ethylenediaminetetraacetic acid (EDTA) and reactivated by the addition of Zn2+. Hydrolysis of poly U by the nuclease showed many intermediate size oligomers prior to the formation of 5'-uridine monophosphate (UMP). Therefore, it was concluded that nuclease Le1 was a Zn(2+)-endonuclease similar to P1-nuclease from Penicillium citrinum. The nuclease was very sensitive to ionic strength, but pH-profiles of the hydrolysis of four 3'-nucleotides were very similar to those of P1 nuclease from P. citrinum.     

Modelling gradient elution of bioactive multicomponent systems in non-linear ion-exchange chromatography

A theoretical framework for the ion-exchange behaviour of bioactive substances in non-linear ion-exchange chromatogaphy is described. The aim of the study was the creation of a model basis to support a process design for production-scale ion-exchange chromatography. The theory can be applied to a whole variety of biological substances, such as amino acids, polysaccharides, peptides and proteins and either isocratic or gradient elution can be carried out. The influence of the eluent concentration on the ion-exchange as well as on the characteristic charge was considered. Experimental measurements showed a strong non-linear ion-exchange equilibrium with a transition from a Langmuir-type to a sigmoidal isotherm at higher eluent concentrations. Hereby, the compound binds to the surface though it is not ionic. Therefore, the model considered the possibility of ion-exchange as well as adsorption. A simplified distribution of the counter-ions based on the Gouy-Chapman theory with a discrete distribution of the counter-ions was used. The theory was extended by a selectivity in the double layer to allow specific adsorption. Calculations of adsorption-elution cycles showed, in agreement with the experimental observations, the development of non-linear elution profiles with a desorption fronting. As a result, the column loading and the eluent concentration were varied. The effect of contaminants, in this case sodium ions, was investigated and included in the model. Finally, the model was extended to multicomponent systems to investigate the effect of side components on the retention behaviour. The development of the characteristic elution profiles and the effect of the column loading on the separation are discussed. Calculated concentration profiles along the column at discrete time steps were used to reveal the influence of side components and the underlying separation mechanism. The simulations provided a new insight into the phenomena involved in biochromatography and make convenient design concepts at least doubtful as the separation is in this case mainly determined by the loading step and not by the choice of the elution gradient.     

Selective elution of soluble rat liver glutathione transferases from a glutathione-Sepharose affinity column

Glutathione transferases (GST) are dimeric enzymes that take part in many detoxification processes. A previous report described the use of a glutathione-Sepharose affinity matrix for the purification of human liver GST. The method involved the use of 5 mM glutathione in a high pH buffer, and the yields were nearly 100%. This method and adapted techniques have now been applied to rat liver GST. Selective GST elution can be obtained in several different ways: by stepwise change of the pH and/or glutathione concentration, and by linear gradient elution. Gel electrophoresis showed, however, that none of the fractions contained pure GST isoenzymes. Also, less than 50% of the total rat liver GST was eluted with 5 mM glutathione, in contrast to the results with human liver GST. A glutathione concentration of 30 mM is necessary for quantitative desorption of rat liver GST from a glutathione-Sepharose column.     

Influence of column design on process-scale ion-exchange chromatography

The performance of two new designs of pump-packed axial flow process chromatography columns have been evaluated for the preparative anion-exchange chromatography of hen egg-white proteins using Whatman Express-Ion Exchanger Q. A 16 1 Side-Pack column and a 24 1 IsoPak column containing Express-Ion Q were used in this study. In each case ca. 20 1 feedstock containing 5-7 g protein/l, was applied per litre packed bed at flow-rates of ca. 150 and 300 cm/h. In each case the ovalbumin binding capacity was ca. 70 g/l packed bed with ca. 100% (w/w) recovery of applied protein. A clean-in-place procedure involving storage in 0.5 M NaOH was effective in maintaining chromatographic performance in all cases. These data were consistent with our previous work using the more traditionally configured slurry-packed axial flow columns. Each of these column designs were easy to use facilitating rapid packing with this adsorbent and in the case of IsoPak rapid pump unpacking. The introduction of these column designs significantly improves the task of column packing, hitherto a labour intensive, physically demanding and potentially unreproducible process.     

An instance of temperature-dependent elution order of enantiomers from a chiral brush-type HPLC column

Typically, reduction of column temperature increases the enantioselectivity of a chiral stationary phase. An instance in which progressive reduction of temperature initially reduces enantioselectivity, then inverts the elution order of the enantiomers, and finally causes enantioselectivity to increase has been observed. This behavior is related to the nature and concentration of the polar modifier in the mobile phase, and requires particular chiral phase-analyte-mobile phase combinations. A rationalization is presented as to the possible origin of this behavior. This rationale may aid in finding other examples of this temperature-dependent elution order of enantiomers and ultimately increase our understanding of chiral recognition processes.     

Purification of S1 nuclease from Aspergillus oryzae by recycling isoelectric focusing

We describe the purification of a single-strand nuclease from Aspergillus oryzae using the first commercial prototype of an instrument (RF3TM) designed by Milan Bier et al. for preparative-scale isoelectric focusing. Protein separation takes place entirely in solution, with shear-stabilized laminar flow counteracting convective disturbances generated by the electric field. Conditions for isoelectric focusing were determined by focusing fractions with nuclease activity, following chromatography on DEAE-Sepharose, in analytical gels containing carrier ampholytes. The separation was then scaled up to process larger quantities of protein in the RF3. When partially-purified protein (250 mg, 6700 U/mg) was focused in pH 3-4 carrier ampholytes. 67% of the activity was recovered in pooled peak fractions with a specific activity of 54,000 U/mg protein. Overall, 82% of the activity loaded on the RF3 was recovered. Eliminating two steps prior to isoelectric focusing, and increasing the protein load from 250 mg to 1.2 g, produced an enzyme with a nearly four-fold increase in specific activity (from 4000 U/mg protein to 15,000 U/mg protein) but with unacceptable color. Our results indicate that a high quality enzyme can be prepared in quantity when heat denaturation and ammonium sulfate precipitation are included prior to isoelectric focusing.     

Prediction of protein retention at gradient elution conditions in ion-exchange chromatography.

This work presents a prediction procedure for protein retention in ion-exchange chromatography, where two linear gradient experiments of different length give the protein retention time at other linear gradients. The procedure predicts the retention time of early and late eluting proteins with similar precision and predictions by extrapolation deviate approximately 3% or less from the experimental retention times. By using the ionic strength, this procedure predicts protein retention times obtained with divalent ions in the eluent more accurately than a well-established procedure that uses the protein co-ion concentration.     

Separation and preconcentration of the rare-earth elements and yttrium from geological materials by ion-exchange and sequential acid elution

The abundance of rare-earth elements (REE) and yttrium in geological materials is generally low, and most samples contain elements that interfere in the determination of the REE and Y, so a separation and/or preconcentration step is often necessary. This is often achieved by ion-exchange chromatography with either nitric or hydrochloric acid. It is advantageous, however, to use both acids sequentially. The final solution thus obtained contains only the REE and Y, with minor amounts of Al, Ba, Ca, Sc, Sr and Ti. Elements that potentially interfere, such as Be, Co, Cr, Fe, Mn, Th, U, V and Zr, are virtually eliminated. Inductively-coupled argon plasma atomic-emission spectroscopy can then be used for a final precise and accurate measurement. The method can also be used with other instrumental methods of analysis.     

Purification of serine hydroxymethyltransferase from Bacillus stearothermophilus with ion-exchange high-performance liquid chromatography.

The gene of serine hydroxymethyltransferase (SHMT) of a thermophilic bacterium Bacillus stearothermophilus was expressed in Escherichia coli, and SHMT was successfully purified from the crude extract of E. coli in two steps while maintaining the enzymatic activity. The purification steps involved ammonium sulphate precipitation followed by high-performance liquid chromatographic separation using the anion-exchange column Fractogel EMD DEAE-650(S). In addition to the DEAE column, three other types of anion- and cation-exchange columns were also studied for their ability to separate SHMT, and the performance of the four columns were compared.     

Purification and characterization of a nuclease (3'-nucleotidase) from a Penicillium sp

A nuclease (3'-nucleotidase) similar to P1 nuclease from Penicillium citrinum was purified from a commercial digestive from a Penicillium sp. The activity of the nuclease (PA) was separated to three fractions by diethylaminoethyl-Toyopearl 650M column chromatography, in total yield of 10%. The apparent molecular weight of these three nucleases, PA1, PA2 and PA3 was 35000, 33000, and 32000, respectively. All of them were homogeneous so far as checked by sodium dodecyl sulfate slab gel electrophoresis. The three nucleases differed in carbohydrate content, but their amino acid composition was practically the same, and very similar to that of P1 nuclease. The molecular weight of nuclease PA3, the major component of nuclease PA, was approximately 27000 after digestion by endoglycosidase F. The N-terminal and C-terminal amino acid sequences of nuclease PA3 were determined by Edman degradation and carboxypeptidase(s) digestion, respectively. The nuclease PA3 was inactivated in the presence of 10 mM ethylenediamine tetraacetic acid (EDTA) and 65% of its native enzyme activity restored by the addition of 20 mM ZnCl2. The pH-dependent photooxidative inactivation of nuclease PA3 was accelerated by removal of Zn ion by EDTA or trishydroxymethyl aminomethane, indicating the possible chelation of Zn2+ with some histidine residues.     

Purification of synthetic oligodeoxyribonucleotides by ion-exchange high-performance liquid chromatography

Synthetic oligodeoxyribonucleotides ranging from 11 to 37 nucleotides in length and with varying base compositions, prepared by both the phosphotriester and phosphite procedures, have been purified by ion-exchange high-performance liquid chromatography on Whatman Partisil 10/SAX columns using phosphate buffer gradients. The effects of different buffer systems on elution times and resolution have been evaluated. Oligomer composition and length had a marked effect on the resolution achieved. In general the use of formamide buffers gave the best results, particularly in the case of 2'-deoxyguanosine-rich sequences. These methods have also been successfully applied to the purification of mixtures of synthetic oligodeoxynucleotides.     

Computer simulation of ion chromatography separation: an algorithm enabling continuous monitoring of anion distribution on an ion-exchange chromatography column

A computer algorithm for the calculation of ion chromatography separation is presented. It is based on the calculation of equilibrium concentrations of present analyte in discrete column segments. The continuous column is treated as a number of discrete cells or segments where the equilibration process between the stationary phase and the eluent is simulated. The ion-exchange equilibration process is supposed to be instantaneous and quantitative. The continuous flow of the eluent is rendered by discrete transfers. The size of each transfer of the eluent corresponds to a portion of the volume contained in one column segment. The equilibrium calculations in all column segments are repeated for each transfer of the eluent, through all the stages of the chromatographic process. The distribution of the analytes between the stationary phase and the eluent can be monitored at any step and in any column segment which means that the described algorithm provides the spatial and time concentration profiles. The simulated chromatogram is acquired as a time-concentration profile in the last column segment. The obtained chromatograms are in good agreement with the experimental ones. The distribution of ions between the stationary phase and the eluent in the early stages of the ion chromatographic process can thus be studied with confidence.     

High-performance ion-exchange chromatography of myosin using a DEAE-5PW column

High-performance ion-exchange chromatography of myosin using a DEAE-5PW packing was used to purify myosin from skeletal, cardiac and smooth muscle. This method produces high-speed resolution (30-min analysis) of myosin from contaminating myofibrillar proteins. The column has a high capacity for binding myosin (up to 1 g) and can be used for small-scale preparation of highly purified myosin. Gel analysis in the presence of sodium dodecyl sulfate showed recovery of myosin with very little contamination of other myofibrillar proteins. Myosin was also recovered from small biopsy samples (0.1 g) by a direct extraction technique with recovery of biological ATPase activity.