Chromatographic separations of serum proteins on immobilized metal ion stationary phases

The separation of proteins on stationary phases consisting of a bound organic chelator and a chelated divalent transition metal has been studied as a function of (A) metal ion species; (B) mobile phase composition and pH; and (C) anion and cation concentration. Optimum separation was observed at alkaline pH on chelated nickel stationary phases. Ammonium and Tris salts reduced the affinity of the metal chelate packing for serum proteins. Halide ions caused the proteins to be more strongly bound to the stationary phase. High salt concentrations had only a small effect on the binding of serum proteins in the absence of amine containing buffers or salts. It was also observed that the ease of elution and the recovery of protein were dependent on pH and upon the presence of halides. The general order of elution of serum proteins, based on isoelectric focusing, was independent of metal ion species and elution conditions, suggesting that a single mechanism or a unique sequence of mechanisms was operative. The results suggest that ligand exchange is the major mechanism of separation under basic conditions and that hydrophobic effects are the result of the competition of nonnitrogen ions with ammonium ions or amines for ligand binding sites modifying or participating in protein binding. Protein binding studies under weak acidic conditions are also presented although the mechanism responsible for protein binding is unclear.     

Separation of both fibrous and globular proteins on the basis of molecular weight using high-performance size exclusion chromatography

A high-performance size exclusion liquid chromatographic system has been used to separate proteins with different shapes solely on the basis of their molecular weights. After the effects of ionic and hydrophobic interactions with the stationary phase have been overcome, protein elution is normally governed by their effective size in solution. Conditions are described under which proteins, with isoelectric points within the normal operating pH range of the columns, are eluted independent of their Stokes' radii. Even fibrous proteins with axial ratios of 50 elute according to their known molecular weights over the range 2000–2,000,000.     

Development of simple and rapid elution methods for proteins from various affinity beads for their direct MALDI-TOF downstream application

Commercially available desalting techniques, necessary for downstream MALDI-TOF analysis of proteins, are often costly or time consuming for large-scale analysis. Here, we present techniques to elute proteins from various affinity resins, free from salt and ready for MALDI mass spectrometry. We showed that 0.1% TFA in 50% acetonitrile or 40% ethanol can be used as salt-free eluents for His-tagged proteins from variety of polyhistidine-affinity resins, while washing of resin beads twice with double-distilled water prior to the elution effectively desalted and recovered wide-range-molecular size proteins than commercially available desalting devices. Modified desalting and elution techniques were also applied for Flag- and Myc-tag affinity resins. The technique was further applied in co-precipitation assay, where the maximum recovery of wide-range molecular size proteins is crucial. Further, results showed that simple washing of the beads with double distilled water followed by elution with acetonitrile effectively desalted and recovered 150 kDa factor H protein of the sheep and its binding partner ~30 kDa BbCRASP-1 in co-precipitation assay. In summary, simple modifications in the desalting and elution strategy save time, labor and cost of the protein preparation for MALDI mass spectrometry; and large-scale protein purifications or co-precipitations can be performed with ease.     

Immobilized metal ion affinity chromatography

This article describes the technique of immobilized metal ion affinity chromatography (1MAC). The IMAC stationary phases are designed to chelate certain metal ions that have selectivity for specific groups in peptides and on protein surfaces. The number of stationary phases that can be synthesized for efficient chclation of metal ions is unlimited, but the critical consideration is that there is enough exposure of the metal ion to interact with the proteins, preferably in a biospecific manner. The versatility of IMAC is one of its greatest assets. An important contribution to the correct use of IMAC for protein purification is a simplified presentation of the various sample elution procedures.     

Predicting the elution behavior of proteins in affinity chromatography on non-porous particles.

Affinity chromatography on non-porous particles of microsize is particularly useful for the rapid analysis and micropreparative separation of proteins. The elution behavior of proteins in an affinity column packed with non-porous copolymerized particles of styrene, methyl methacrylate and glycidyl methacrylate was investigated both theoretically and experimentally, using the lysozyme-Cibacron Blue 3G-A affinity system. Equations used to predict the elution profiles, resulting from the elution by increasing the ionic strength (NaCl concentration) in the mobile phase, were obtained. The maximum adsorbate concentration, desorption rate constant and equilibrium constant under elution conditions were determined by matching experimental data with predicted elution profiles. Based on the parameters determined at a flow-rate of 0.5 ml/min and with 1 M NaCl in the elution buffer, the model equations could predict the elution profiles for other experimental runs, where different flow-rates and sodium chloride concentrations were used. Both the experimental and predicted results revealed that the affinity interaction kinetics are not significantly influenced by the flow-rate and, hence, the film mass transfer. To elute bound lysozyme from immobilized dye ligand, a higher value of the ionic strength leads to a faster elution and a sharper elution peak. The influence of elution conditions on the kinetic and thermodynamic parameters and, consequently, on the elution peak profiles was evaluated. The model equations can also predict the behavior of protein elution from an affinity column by changing the pH of the mobile phase, according to a previous study.     

Additive concentration effects on enantioselective separations in supercritical fluid chromatography

Polar additive concentration effects in supercritical fluid chromatography were studied on chiral stationary phases having either a macrocyclic glycopeptide or a derivatized polysaccharide as the chiral selector. Two basic additives, isopropylamine and triethylamine, were incorporated into the methanol modifier at various concentrations and the effects on retention, selectivity, and resolution were monitored. Many of the analytes failed to elute from the macrocyclic glycopeptide stationary phase in the absence of an additive and the most noticeable effect of increasing additive concentration was a significant decrease in retention. On the derivatized polysaccharide stationary phase the additives had little effect on retention, but they did foster significant improvements in peak shape and resolution.     

Design of affinity tags for one-step protein purification from immobilized zinc columns

Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to be superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. For example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper we have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.     

Species-dependent binding of serum albumins to the streptococcal receptor protein G

The interaction of the serum albumin binding domain from streptococcal protein G to serum albumins isolated from different species was investigated. The highest affinity to protein G was found for serum albumins from rat, man and mouse. A medium binding was found for serum albumin from rabbit, cow, hen and horse, while little or no binding was found for ovalbumin and serum albumin from sheep. The interaction between human serum albumin and protein G showed rapid binding kinetics at the temperatures 7, 22 and 37 degrees C. Furthermore, the ability of different serum albumins to function as affinity ligands when covalently coupled to a solid support was tested. The results show that protein G derivatives could be eluted at different pH depending on the origin of the serum albumin. It was also possible to elute the streptococcal receptor efficiently from the mouse serum albumin matrix with human serum albumin. Based on these results, a gene fusion system for recovery of sensitive proteins by affinity purification is described, where high yields are obtained under mild elution conditions.     

high-performance liquid chromatography of amino acids, peptides and proteins : LXIII. Reversed-phase high-performance liquid chromatographic characterisation of several polypeptide and protein hormones

The chromatographic behaviour on alkylsilicas of a variety of hormonal proteins is described. Optimization of resolution and recovery of these protein hormones, which included porcine relaxins, human chorionic gonadotropin, human placental lactogen, pituitary derived growth hormone and adenohypophyseal glycoprotein hormones, was achieved by manipulation of both mobile and stationary phase parameters. With standard stainless-steel analytical columns (10–30 cm × 0.4 cm) packed with meso- or macro-porous n-alkylsilica supports these proteins can be readily fractionated at the semi-preparative level with separation times generally under 90 min using elution systems directly compatible with subsequent methods of primary structure determination or biological functional analysis. The effects of changes in several experimental parameters on peak symmetry, retention and recovery are described.     

The interaction of proteins with hydroxyapatite. I. Role of protein charge and structure

The criteria for elution of proteins from hydroxyapatite columns were examined as a function of (1) protein isoelectric point (22 proteins with isoelectric points between 3.5 and 11.0); (2) ionic nature of eluant (Na salts of PO4, F-, Cl-, SCN-, ClO-4, and CaCl2); and (3) structural differences between related proteins. It was found that proteins can be classified into three groups: (1) basic proteins, which elute at similar, moderate molarities of PO4, F-, Cl-, SCN-, and ClO-4, and low (less than 0.003 M) Ca2+; (2) acidic proteins which elute at about equal moderate molarities of PO4 and F-, but do not elute with Ca2+ and usually not with Cl-; (3) neutral proteins, which elute with PO4, F-, and Cl-, but show a strong anion specificity, and do not elute with Ca2+ or SCN-. Furthermore, individual specific polar groups are not in general crucial to binding or desorption, and variations in structure, other than major loosening, do not influence strongly the pattern of protein-hydroxyapatite interaction.     

Use of cellulose-based stationary phases for chiral separation in open tubular column chromatography

Application of cellulose-based chiral stationary phases was extended to open tubular columns. These chiral materials were mixed with achiral matrix stationary phases. Compromises were found among the polarity and the ratio of achiral matrix polymers against the content of the chiral cellulose derivative in order to optimize the resolution of the investigated racemates. In GC, the high efficiency feature of open tubular columns allows fast analysis, however, compounds which express strong H-bond interaction with cellulose derivatives elute with a bad peak shape. The application of these stationary phases for open tubular SFC was more successful, because the solvation power of the mobile phase can compensate the strong interaction between the solute and the cellulose derivative. Immobilization of the stationary phases were achieved for SFC purposes. © 1992 Wiley-Liss, Inc.     

Improved performance of column chromatography by arginine: dye-affinity chromatography

Arginine has been effectively used in various column chromatographies for improving recovery and resolution, and suppressing aggregation. Here, we have tested the effectiveness of arginine as an eluent in dye-affinity column chromatography using Blue-Sepharose, which binds enzymes requiring adenyl-containing cofactors (e.g., NAD). A common eluent, NaCl, showed a broad elution peak with low recovery of lactate dehydrogenase, at most approximately 60% using 2M salt. The recovery decreased as the NaCl concentration was either decreased or increased; i.e., the recovery was maximum at 2M. On the contrary, addition of arginine to the eluent resulted in more than 80% recovery above 0.5M and the recovery was nearly independent of the arginine concentration. The elution peak was much sharper with arginine, leading to elution of more concentrated protein solution. Successful elution of proteins bound to the ATP-agarose resins by arginine was also described.     

Retention behaviour of proteins on poly(vinylimidazole)-copper(II) complexes supported on silica: application to the fractionation of desialylated human α1-acid glycoprotein variants

The retention behaviour of various amino acids, peptides and proteins on poly(vinylimidazole)-Cu(II) complexes supported on silica was investigated. Free amino acids and peptides containing one histidine and in some instances one additional tryptophan residue in their primary structure were found to elute from the supports only after addition of a competing complexing agent to the mobile phase. However, the results obtained with proteins containing metal binding groups suggested that, in addition to the presence of donor-acceptor interactions between the macromolecules and the immobilized metal, other additional (essentially ionic and/or hydrophobic) interactions took place between the proteins and the surrounding of the metal. When donor-acceptor interactions were predominant, proteins were strongly adsorbed on the stationary phase and their elution required the addition of a competing complexing agent in the mobile phase. However, when the binding between the proteins and the supports via donor-acceptor interactions was less favourable, proteins were eluted from the columns without the addition of a competing agent in the mobile phase. With respect to the binding of these proteins, ionic and/or hydrophobic interactions were no longer negligible during the chromatographic process and the retention of the macromolecules by the stationary phase depended on the elution conditions (ionic strength, pH, etc.). These supports were used in the fractionation of the three main genetic variants of desialylated α₁-acid glycoprotein.     

New internal standards for basic amino acid analyses

Eight derivatives of cysteine and penicillamine with 2- and 4-vinyl-pyridine, p-nitrobenzyl bromide, and p-nitrostyrene were evaluated as potential internal standards for the short and long (physiological) basic columns in amino acid analysis by ion-exchange chromatography. S-β-(4-pyridylethyl)-dl-penicillamine (4-PEP) was found to have an advantage over the previously proposed S-β-(4-pyridylethyl)-l-cysteine (4-PEC) since the elution position of 4-PEP on the short basic column is insensitive to minor changes in pH of the eluting citrate buffer. 4-PEP was found to be stable to acid hydrolysis as used for proteins and its recovery from protein hydrolysates was unaffected by the presence of starch during hydrolysis. However, an extra 14 min is required to elute 4-PEP on the short column.Of the eight compounds studied, six appcar suitable as internal standards on the physiological (long) column. These elute in widely differing positions between histidine and arginine, thus offering a choice of internal standards for special analysis on the basic long column.     

Refolding strategies for ketosteroid isomerase following insoluble expression in Escherichia coli

Dilution and column-based protein refolding techniques are compared for refolding Delta 5-3-ketosteroid isomerase (KSI) with a C-terminus his6-tag. Column refolding was performed by removing the denaturant while the protein was adsorbed in an immobilized metal affinity chromatography column. Both dilution refolding and a single-step column-based refolding strategy were optimized to maximize the recovery of KSI enzyme activity, and achieved refolding yields of 87% and 70% respectively. It was found that the column-based refolding yield was reduced at higher adsorbed protein concentrations. An elution gradient with increasing imidazole concentration was used to selectively elute the biologically active KSI protein following column refolding, with high molecular weight KSI aggregates retained in the column. An iterative column-refolding process was then developed to denature and refold protein retained in the column, which significantly increased the refolding yield at high-adsorbed protein concentrations. Repetition of the column refolding operation increased the refolding yield from 50% to 75% for protein adsorbed at a concentration of 2.9 mg/mL of adsorbent. Although for the KSI protein column-based refolding did not improve the overall refolding yield compared to dilution refolding, it may still be advantageous due to the ease of integration with purification operations, increased control over the refolding conditions, and the ability to segregate refolded protein from inactive aggregates during elution.     

Two step capture and purification of IgG2 using multicolumn countercurrent solvent gradient purification (MCSGP)

A two‐step chromatography process for monoclonal antibody (mAb) purification from clarified cell culture supernatant (cCCS) was developed using cation exchange Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) as a capture step. After an initial characterization of the cell culture supernatant the capture step was designed from a batch gradient elution chromatogram. A variety of chromatographic materials was screened for polishing of the MCSGP‐captured material in batch mode. Using multi‐modal anion exchange in bind‐elute mode, mAb was produced consistently within the purity specification. The benchmark was a state‐of‐the‐art 3‐step chromatographic process based on protein A, anion and cation exchange stationary phases. The performance of the developed 2‐step process was compared to this process in terms of purity, yield, productivity and buffer consumption. Finally, the potential of the MCSGP process was investigated by comparing its performance to that of a classical batch process that used the same stationary phase. Biotechnol. Bioeng. 2010;107: 974–984. © 2010 Wiley Periodicals, Inc.     

Fractionation of DNA from mammalian cells by alkaline elution.

The method of alkaline elution provides a sensitive measure of DNA single-strand length distribution in mamalian cells and is applicable to a variety of problems concerning DNA damage, repair, and replication. The physical basis of the elution process was studied. The kinetics of elution above the alkaline transition pH were found to occur in two phases: an initial phase in which single-strand length is rate limiting, followed by a phase in which elution is accelerated due to the accumulation of alkali-induced strand breaks. The range of DNA single-strand lengths that can be discriminated by elution above the alkaline transition pH was estimated by calibration relative to the effects of x ray, and was found to be 5 X 10(8)-10(10) daltons. Shorter DNA strands elute within the pH transition zone, which extended from pH 11.3 to 11.7 when tetrapropylammonium hydroxide was used as base. This elution was relatively rapid, but was sharply limited by pH, according to the length of the strands: the length of the strands eluted increased with increasing pH. Alkaline elution was inhibited by treatment of cells with low concentrations of nitrogen mustard, a bifunctional alkylating known to cross-link DNA. On investigation of the possibility that DNA subclasses may differ in their elution behavior, satellite L strands were found to elute more slowly from cells exposed to a low dose of x ray than did the bulk DNA.     

Separation of the major proteins of central and peripheral nervous system myelin using reversed-phase high-performance liquid chromatography

A general method to separate the major proteins of rat central and peripheral nervous system myelin has been developed. The key step is the initial quantitative removal of the lipids under conditions where the proteins retain their solubility in HPLC solvents. Lipids are removed by a combination of solvent extraction and column chromatography on Sephadex LH-60 in 2-chloroethanol:10 mM HCl (9:1). Proteins are then separated by reversed-phase (RP) HPLC. Samples are applied to a wide pore reversed-phase C-3 column and eluted with a linear gradient of 10-70% 1-propanol in 0.1% trifluoroacetic acid (0-100% B) over a 60-min period. Myelin basic proteins elute between 25 and 30% B, Wolfgram and other high molecular weight proteins at 35-50% B, proteolipid protein at 65-80% B, and P0 glycoprotein at 55-65% B. This elution pattern is consistent with the known relative hydrophobicity of these proteins. Protein recovery for the entire procedure is greater than 74%. Proteolipid and P0 proteins isolated by HPLC contain 2.3 and 1.1 mol of covalently bound fatty acids, respectively. This fatty acid composition is similar to that previously reported using different isolation procedures. The analysis of central and peripheral nervous system myelin proteins by RP-HPLC permits the isolation of purified proteins for structural and metabolic experiments.     

Capture of a recombinant protein from unclarified canola extract using streamline expanded bed anion exchange

The feasibility of applying expanded bed adsorption technology to recombinant protein recovery from extracts of transgenic canola (rapeseed) was assessed. The extraction step results in a suspension of high solids content that is difficult to clarify. The coarse portion of the solids can be removed easily, and our aim was to operate the expanded bed in the presence of the recalcitrant particulates. Recombinant beta-glucuronidase (rGUS) produced in transgenic canola seed was the model system. Diethylaminoethyl (DEAE) and Streamline DEAE resin exhibited similar binding and elution properties for both rGUS and native canola proteins. More than 95% of native canola proteins did not bind to DEAE resins at pH 7.5, whereas the bound proteins were fractionated by two-step salt elution into two groups with the first peak, containing 70% of total bound proteins, at 20 mS/cm, followed by elution of rGUS at 50 mS/cm. The adsorption isotherm was only slightly influenced by the presence of up to 14 mg solids/mL extract; C(m) and K(d) changed by -1% and +39%, respectively. Bed expansion was semiquantitatively predictable from physical properties of the fluid together with Stokes's law and the Richardson-Zaki correlation for both clarified and partially clarified extracts. The presence of 1.4% solids did not change rGUS breakthrough behavior of the expanded bed; however, a small difference between expanded bed and packed bed was observed early in the sample loading stage, during which bed expansion adjusts. Canola solids moved through the column in approximately plug flow with no detriment to bed stability. Seventy-two percent recovery of 34-fold purified rGUS was obtained after initial loading of 1.4% (w/w) solids extract to 25% breakthrough.     

Use of high-performance size-exclusion chromatography to measure protein molecular weight and hydrodynamic radius. An investigation of the properties of the TSK 3000 SW column

We have conducted a study of the TSK 3000 SW high-performance size-exclusion column to define under what conditions proteins would migrate most consistently with their known hydrodynamic properties. Our findings include the following: 1) the residual negative charge of the column does cause charge-exclusion or charge-retention effects at low ionic strengths; with elution in deionized water several anionic proteins elute approximately in the void volume; 2) at mu greater than or equal to 0.5, protein migration is not only independent of ionic strength, but consistent with protein molecular weight and hydrodynamic volume; 3) small hydrophobic peptides are retarded by the column; and 4) very asymmetric proteins and other hydrodynamic particles are likely to be retarded by an "end-on insertion" mechanisms.