Chromatofocusing of peptides and proteins using linear pH gradients formed on strong ion-exchange adsorbents

Although it is commonly believed that a column packing used for chromatofocusing must have an "even" buffering capacity in order to produce a linear pH gradient, it is demonstrated here that linear pH gradients suitable for chromatofocusing can be produced on a column packing having a minimal buffering capacity. In particular, if either a strong-acid cation-exchange column packing or a strong-base anion-exchange column packing is presaturated with either a weak acid titrated with a strong base, or a weak base titrated with a strong acid, respectively, to the initial pH, then a linear or nearly linear pH gradient can be formed using a polyampholyte elution buffer by taking advantage of the presence of small quantities of weak-acid or weak-base functional groups that generally exist on these types of column packings. Experimental and theoretical studies are used to demonstrate that such systems have potential advantages over traditional chromatofocusing methods in terms of the speed of the separation, the resolution achieved, and the range of applications possible. Among other techniques described, a method for separating tryptic peptides using chromatofocusing and a strong-acid cation-exchange column packing is demonstrated to be a useful alternative to capillary isoelectric focusing and ion-exchange chromatography using a salt gradient for this purpose.     

pH Transitions in ion-exchange systems: role in the development of a cation-exchange process for a recombinant protein

Unexpected transient changes in effluent pH can occur during ion-exchange chromatography. Such changes can occur even if a column that is equilibrated with a buffer receives another solution in the same buffer and of the same pH but of a different salt concentration. An attempt is made to understand the basis for this phenomenon and apply it to the process purification of a recombinant protein on a strong cation-exchange resin. Incomplete column equilibration was eliminated as a possible cause of these effects. Various buffering species and various salt ions were studied at different solution concentrations to investigate pH transitions on strong cation-exchange resins. A further comparison was made between cation-exchange resins with different backbone chemistries. On the basis of these studies, a mechanism is proposed for these phenomena based on competitive equilibria between ions from the buffer salts and H(+)/OH(-) ions. In addition to the equilibria between these ions and the functional groups on the resins, charged groups on the resin backbone were also found to contribute to transient pH changes. The results from this study were applied to the cation-exchange step for a recombinant protein that was sensitive to pH excursions to help maintain activity of the protein during the purification process.     

Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography

In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%-60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.     

Analysis of rat uterine estrogen receptors by high-pressure liquid chromatography methods

Cytosolic (ERc) and nuclear (ERn) estrogen receptors prepared from rat uteri were characterized by size-exclusion and ion-exchange HPLC. The oligomeric ERc eluted as a single, sharp peak near the exclusion volume of the gel column; ERn eluted as a broad peak. When salt-extracted ERn was partially purified sequentially by Sephadex G-200, DEAE-cellulose chromatography and polyacrylamide gel electrophoresis, the partially purified receptor moieties were not distinguishable by the sucrose gradient method, but showed characteristic retention times in the size-exclusion HPLC column. Further distinction in net surface charges was observed between ERc and ERn moieties by ion-exchange high-pressure liquid chromatography (HPLC). Molybdate-stabilized ERc was eluted as sharp peak at 0.27 M salt gradient. In contrast, fresh extracts of ERn emerged as a broad peak in the region of 0.1-0.2 M salt gradient. In the absence of molybdate, ERc dissociated into several 4-5 S molecules, which were well resolved in the DEAE column. This report, therefore, demonstrates the usefulness of size-exclusion and ion-exchange HPLC for steroid receptor analysis.     

Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography

In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%–60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.     

Displacement effects in large-scale chromatography?

Displacement effects in large-scale (total column volume v(t) = 150 L) and preparative ion-exchange chromatography purifying human erythrocyte superoxide dismutase are described in the present article. The biomolecules are eluted in a very small peak elution volume (<0.2 v(o)) behind the salt wave using a step gradient. The theoretical peak width and retention behavior are calculated according to the model of Yamamoto. The theoretical values are then compared with the experimental data. There was a difference observed between the elution type I (also called fronting) and the experimentally obtained elution. Some instructions are given on how to achieve these phenomenona because a beneficial effect in respect to resolution and recovery of a biomolecule is observed.     

普通离子交换剂用于HPLC柱层析法快速分离眼镜王蛇毒

目的　为了经济快速分离眼镜王蛇(Ophiophagushannah,Oh)蛇毒中的毒素成分。　方法　用普通离子交换剂于高效液相色谱柱 (HPLC) TSKgel SP-Toyopearl 65 0 SF (4× 1 5 0 mm)层析法 ,实验取得最佳分离条件后 ,将蛇毒样品上柱后进行梯度洗脱 ,各洗脱峰收集后在 Cosmosil 5 C4-AR-3 0 0柱 (4 .6× 1 5 0 mm)上进行逆相 HPLC分析。非单峰组分再进行 HPLC凝胶过滤柱TSKgel Toyopearl HW-40 Fine(4× 2 5 0 mm)层析 ,层析峰组分再进行 HPLC逆相分析。　结果　眼镜王蛇毒经HPLC离子交换柱层析获得了 1 6个蛋白组分 ,其中有 5个组分经逆相 HPLC分析单一组分 ;另外的复合性组分再进行 HPLC凝胶过滤柱层析后又得到 5个单峰蛋白组分。　结论　HPLC离子交换柱层析对分离蛇毒蛋白很有实用价值 ,特别是蛇毒样品量少的情况下 (1 0 ug)也能较好分离。还具有分离时间短 (1 h左右 ) ,无须低温条件等优点。HPLC凝胶过滤柱层析可进一步使蛋白组分得到提纯     

Purification of recombinant green fluorescent protein using chromatofocusing with a pH gradient composed of multiple stepwise fronts

Green fluorescent protein (GFP), which fluoresces in the green region of the visible spectrum and is widely used as a reporter for gene expression and regulation, was overexpressed in the JM105 strain of Escherichia coli transformed with pBAD-GFP. A two-step chromatofocusing procedure was used to purify GFP starting from cell lysate, with each step employing a pH gradient extending from pH 5.5 to 4.0. The first chromatofocusing step was performed using a low-pressure column in which a retained stepwise pH front formed by adsorbed buffering species was used to capture GFP directly from clarified cell lysate and selectively focus it into a chromatographic band. The second step utilized a high-performance column under mass overloaded conditions where a similar pH front acted as a protein displacer and led to the formation of a highly concentrated rectangular band of GFP. The overall procedure yielded a 50-fold increase in purity, a 20-fold volume reduction, and a recovery and purity for GFP of 60% and 80%, respectively. Because the method employs a strong-base ion-exchange column packing and low-cost buffers formed with formic and acetic acids instead of the proprietary column packings and polyampholyte elution buffers more generally used for chromatofocusing, it appears to be a practical alternative for the preparative ion-exchange chromatography of GFP in particular and for the recovery of recombinant proteins from cell lysate in general. A discussion is also given concerning the choice of appropriate buffers for the rational design of pH gradients involving retained, stepwise pH fronts that span a given pH range and of the use of the fluorescence properties of GFP for flow visualization and chromatographic process development.     

Purification of Escherichia coli alkaline phosphatase on an ion-exchange high-performance liquid chromatographic column using carboxymethyl dextrans

Carboxymethyl dextrans (CM-Ds) were used on an HPLC ion-exchange column to obtain significantly enriched alkaline phosphatase (EC 3.1.3.1) from a sample of Escherichia coli periplasmic space proteins without significant loss of enzymatic activity. The ability of CM-Ds to separate alkaline phosphatase even when the column was 80-85% saturated with protein demonstrates the potential for high column capacity using CM-Ds. In addition, the fractions containing alkaline phosphatase and CM-Ds were reapplied to the same ion-exchange column under different buffer conditions and purified to homogeneity by salt gradient elution chromatography, thus demonstrating the compatibility of CM-Ds with the latter chromatographic method. The two-step chromatographic procedure yielded enzyme of purity comparable to that of electrophoretically purified E. coli alkaline phosphatase obtained commercially. These studies demonstrate that HPLC displacement chromatography is a mild procedure which allows rapid, quantitative purification of an enzyme. Scaling up with larger columns should allow purification of enzymes of a commercial basis.     

Characterization of uterine estrogen receptors by size-exclusion and ion-exchange high-performance liquid chromatography

This report presents the first application of ion-exchange high-performance liquid chromatography in the study of ER from the rabbit uterus. In the presence of sodium molybdate (20 mM), native ER was eluted as a sharp peak at 0.29 M NaCl by a linear salt gradient, but without molybdate, it resolved into 4 major peaks. Molybdate-stabilized ER from the DEAE column, similar to ER from crude cytosol, sedimented at the 6-8S region in low salt and 4S region in high salt linear sucrose gradients, and was excluded from size-exclusion HPLC. In contrast, dissociated ER subunits from DEAE eluates ranged from 3.5 to 4.5S, and showed differences in molecular weights in a size-exclusion column. These results show that the native ER is a large molecule which dissociates into smaller subunits in the absence of molybdate; each of the steroid-bound moieties differs in molecular weight and surface charge from the native molecule.     

Purification of bacterial L-methionine gamma-lyase

A chromatographic procedure using sequential ion-exchange columns is described for separating choline, trimethylamine, trimethylamine oxide, and betaine extracted from marine fish tissues; added exogenous carnitine can also be separated by the system. Choline with its positive charge binds to the AG 50W-X8 (Na+, pH 9) column. The column is first eluted with 0.1 N NaOH to collect trimethylamine, trimethylamine oxide, and betaine; choline is then eluted with 0.5 N NaOH. The amines collected with 0.1 N NaOH are subsequently separated using an AG 50W-X8 (H+, pH 4) column eluted with a linear 0-1 M NaC1 gradient.     

Isolation and characterization of the K5-type yeast killer protein and its homology with an exo-beta-1,3-glucanase

K5-type yeast killer protein in the culture supernatant of Pichia anomala NCYC 434 cells was concentrated by ultrafiltration and purified to homogeneity by ion-exchange chromatography with a POROS HQ/M column followed by gel filtration with a TSK G2000SW column. The protein migrated as a single band on discontinuous gradient SDS-PAGE and had a molecular mass of 49,000 Da. The pI value of the K5-type killer protein was measured at pH 3.7 by high voltage vertical gel electrofocusing. The result of an enzyme immuno assay revealed that it was a glycosylated protein. Its internal amino acid sequencing yielded the sequences LNDFWQQGYHNL, IPIGYWAFQLLDNDPY, and YGGSDYGDVVIGIELL, which are 100% identical to exo-beta-1,3-glucanase (accession no. AJ222862) of Pichia anomala (strain K). The purified protein was highly stable at pH values between 3 and 5.5 and temperatures up to 37 degrees C.     

Extension of the selection of protein chromatography and the rate model to affinity chromatography

The rational selection of optimal protein purification sequences, as well as mathematical models that simulate and allow optimization of chromatographic protein purification processes have been developed for purification procedures such as ion-exchange, hydrophobic interaction and gel filtration chromatography. This paper investigates the extension of such analysis to affinity chromatography both in the selection of chromatographic processes and in the use of the rate model for mathematical modelling and simulation. Two affinity systems were used: Blue Sepharose and Protein A. The extension of the theory developed previously for ion-exchange and HIC chromatography to affinity separations is analyzed in this paper. For the selection of operations two algorithms are used. In the first, the value of η, which corresponds to the efficiency (resolution) of the actual chromatography and, Σ, which determines the amount of a particular contaminant eliminated after each separation step, which determines the purity, have to be determined. It was found that the value of both these parameters is not generic for affinity separations but will depend on the type of affinity system used and will have to be determined on a case by case basis. With Blue Sepharose a salt gradient was used and with Protein A, a pH gradient. Parameters were determined with individual proteins and simulations of the protein mixtures were done. This approach allows investigation of chromatographic protein purification in a holistic manner that includes ion-exchange, HIC, gel filtration and affinity separations for the first time.     

Isolation and characterization of individual tRNA Leu 1,2,and 4 from the bovine udder

tRNA-Leu 1, tRNA-Leu 2 and tRNA-Leu 4 were isolated from a lactating cow mammary gland by combination of several chromatographic methods. Chromatography on a Sepharose 4B column with a reverse salt gradient was used as the first step. Individual tRNAsLeu were further purified by RPC-5 column chromatography at pH 4.5 and 7.5. For isolation of tRNA-Leu 2 a RPC-5 column was additionally used at pH 3.3 in the presence of 7 M urea. Using micro-column chromatography of Ti-RNAases digests, it was demonstrated that tRNA-Leu 1 and tRNA-Leu 2+ are similar in their primary structure and differ essentially from tRNA-Leu 4.     

Plate height determination for gradient elution chromatography of proteins

A method for determining the plate height HETP from the elution curve obtained by the linear gradient elution (LGE) ion-exchange chromatography (IEC) of proteins is presented. The method was developed on the basis of the numerical solutions of a chromatography model which considers the zone sharpening and the distribution coefficient as a function of the salt concentration. The plate height HETP is determined from the peak width and the salt concentration at which the peak is eluted in LGE. The method was applied to the experimental results with various ion-exchange chromatography media. A calculation example based onthe present method is presented to show how the chromatographic and operating parameters should be tuned to obtain a desired resolution. A simplified calculation procedure for the peak profile is also described. (c) 1995 John Wiley & Sons, Inc.     

Comparison of fractionation strategies for offline two-dimensional liquid chromatography tandem mass spectrometry analysis of proteins from mouse adipose tissue

In the frame of protein identification from mouse adipose tissue, two strategies were compared for the offline elution of peptides from a strong cation exchange (SCX) column in two-dimensional liquid chromatography tandem mass spectrometry (2D–LC–MS/MS) analyses. First, the salt gradient (using K⁺ as displacing agent) was evaluated from 25 to 500 mM KCl. Then, a less investigated elution mode using a pH gradient (using citric acid and ammonium hydroxide) was carried out from pH 2.5 to 9.0. Equal amounts of peptide digest derived from mouse adipose tissue were loaded onto the SCX column and fractionated according to the two approaches. A total of 15 fractions were collected in two independent experiments for each SCX elution strategy. Then, each fraction was analyzed on a nanoLC–MS/MS platform equipped with a column-switching unit for desalting and enrichment. No substantial differences in peptide quality characteristics (molecular weight, isoelectric point, or GRAVY [grand average of hydropathicity] index distributions) were observed between the two datasets. The pH gradient approach was found to be superior, with 27.5% more unique peptide identifications and 10% more distinct protein identifications compared with the salt-based elution method. In conclusion, our data imply that the pH gradient SCX fractionation is more desirable for proteomics analysis of entire adipose tissue.     

High performance liquid chromatography of nucleotides. Major methods and their development

The separation of mono- and oligonucleotides possibilities by means of high performance ion-exchange, reversed-phase, so-called "ion-pair" and adsorption chromatography are studied. The influence of the eluent composition (solvent, salt) and pH on the retention, selectivity and resolution in reversed-phase and ion-exchange chromatography is investigated. The model of the hydrophobic-pair ion-exchange mechanism of ion-pair chromatography is considered. The conditions for analysis and preparative isolation of a desired component are optimized for selectivity, resolution and throughput. The methods for prediction of the optimal gradient elution program reasonable resolution at the desired retention time and for choosing the guard-column packing material are proposed. A design of the gradient for system and the version of slurry packing method for HPLC prolonged life-time columns are improved. The automatized analytical technique for determination of the oligonucleotide monomeric composition with two coupled microcolumns is described, that involves enzymatic digestion of an oligonucleotide followed by ion-exchange separation of the hydrolysate.     

Nonlinear gradient isotherm parameter estimation for proteins with consideration of salt competition and multiple forms.

Salt gradients in ion-exchange chromatography are routinely used to speed separation of proteins and to concentrate products, but systematic optimization of these gradients requires protein equilibrium data as a function of salt concentration. An understanding of conformational changes, aggregation, and salt effects, which include both competition and affinity modulation, is important for equilibrium isotherm parameter estimation. In this study, gradient elution of bovine serum albumin (BSA) in anion exchange was well predicted by a salt-modulated nonlinear isotherm which considers salt competition. The isotherm was able to predict BSA gradient elution from batch equilibrium data. The same isotherm was also able to predict elution for various gradient slopes when fitted to an intermediate slope gradient experiment. If multiple forms due to aggregation or denaturation exist, isotherm parameters are readily averaged in batch experiments because of the long equilibration times. Similarly, gradient experiments yield averaged parameters because the salt gradient tends to merge the closely eluting forms. However, in isocractic elution, if the reaction rate is not rapid enough to give a merged peak, the estimated isotherm parameters are only fair predictors of gradient behavior and vice versa. Slower flow rates in isocratic elution can help reduce the discrepancy by allowing forms to merge through interconversion. As an alternative to determining averaged parameters, consideration of two binding forms, using VERSE-LC, an advanced rate model, gave good agreement with experimental data over the entire range of salt gradient durations.     

Non-reducing alkaline solubilization and rapid on-column refolding of recombinant prion protein

Mature prion protein (PrP) is a 208-residue polypeptide that contains a single disulfide bond. We report an alternative method to purify recombinant mouse PrP produced in Escherichia coli. Bacterial inclusion bodies were solubilized in a buffer containing 2 M urea at pH 12.5. The solubilized protein was rapidly purified on a nickel affinity column without a chaotrope gradient, followed by ion-exchange chromatography. The yield and purity of PrP produced by this alternative approach was similar to that obtained using a conventional solubilization and on-column refolding protocol. Recombinant PrP produced using the non-reducing purification protocol is properly folded, as determined by circular dichroism, and a competent substrate for amyloid fibril formation, as determined by Thoflavin-T dye binding assays. In summary, this report describes a rapid method for producing properly folded recombinant PrP without reducing agents or a chaotrope gradient.     

Cation-exchange displacement chromatography of proteins with protamine displacers: effect of induced salt gradients.

Protamine was investigated for its utility as a protein displacer in cation-exchange systems. Although the protamine solution contained several variants of the molecule, the high affinity of all of the components in this heterogeneous biopolymer enabled it to act as an efficient protein displacer. To facilitate parameter estimation of the protamine, a preliminary purification was carried out by preparative elution chromatography. Chromatographic parameters of both the feed proteins and protamine displacer were obtained for use in a multicomponent steric mass action ion-exchange displacement model. Model simulations were compared to displacement results under both moderate and intense induced salt gradient conditions. In both cases, excellent agreement was obtained between the displacement experiments and theoretical predictions. In addition, these studies serve to dramatize the importance of induced salt gradients in ion-exchange displacement systems.