Protein Chromatography Research Articles

Collapse of Thermoresponsive Brushes and the Tuning of Protein Adsorption

Protein adsorption onto brush displaying surfaces is strongly affected by collapse, an effect utilized in protein chromatography and in harvesting cell sheets for tissue engineering applications. F...

The Dimeric Structure of the Cpn60.2 Chaperonin of Mycobacterium tuberculosis at 2.8 Å Reveals Possible Modes of Function

Mycobacterium tuberculosis expresses two proteins (Cpn60.1 and Cpn60.2) that belong to the chaperonin (Cpn) family of heat shock proteins. Studies have shown that the two proteins have different functional roles in the bacterial life cycle and that Cpn60.2 is essential for cell viability and may be involved in M. tuberculosis pathogenicity. Cpn60.2 does not form a tetradecameric double ring, which is typical of other Cpns. We have determined the crystal structure of recombinant Cpn60.2 to 2.8 Å resolution by molecular replacement; the asymmetric unit (AU) contains a dimer, which is consistent with size-exclusion high-performance liquid chromatography and dynamic light-scattering measurements of the soluble recombinant protein. However, we suggest that the actual Cpn60.2 dimer may be different from that identified within the AU on the basis of surface contact stability, solvation free-energy gain, and functional aspects. Unlike the dimer found in the AU, which is formed through apical domain interactions, the dimeric form we propose here provides a free apical domain that is required for normal chaperone activity and may be involved in M. tuberculosis association with macrophages and arthrosclerosis plaque formation. Here we describe in detail the structural aspects that lead to Cpn60.2 dimer formation and prevent the formation of heptameric rings and tetradecameric double rings.

Retention times and bandwidths in reversed-phase gradient liquid chromatography of peptides and proteins

Due to dramatic effects of even small changes in mobile phase composition on the retention, separations of high-molecular compounds are very difficult, if possible at all, at isocratic conditions and need gradient elution. The theory of gradient elution for small molecules is well established, however its applications to reversed-phase gradient separations of biopolymers are not straightforward because of specific problems, such as slow diffusion, limited accessibility of the stationary phase for larger molecules, or possible sample conformation changes during the elution. Theoretical prediction of gradient data needs the parameters of model retention equations to be known, which however cannot be determined at isocratic conditions. The present work overviews the attempts at implementation of the conventional gradient theory developed for low-molecular compounds to the description and prediction of gradient separations of peptides and proteins on various types of HPLC columns: conventional analytical columns packed with wide-pore fully porous, fused-core superficially porous and non-porous particles; silica-based monolithic columns and organic-polymer poly(alkylmethacrylate) and poly(styrene-divinylbenzene) monolithic columns in capillary and disc formats. The attention is focused on the determination of the parameters necessary to predict gradient retention times (volumes) and bandwidths using the theoretical model equations. The accuracy of the prediction of protein retention on totally porous columns improves if size exclusion effect is taken into account, but this is not necessary with non-porous or superficially porous particles. Band dispersion effects counteracting band compression in gradient elution depend on the type of column, on the protein and on the gradient volume (steepness) and complicate the prediction of band broadening in gradient chromatography of proteins, however the conventional gradient model can be employed to estimate the effects of changing gradient parameters on the bandwidths, as well as on the elution times (volumes) of proteins.

Protein adsorption and transport in polymer-functionalized ion-exchangers.

A wide variety of stationary phases is available for use in preparative chromatography of proteins, covering different base matrices, pore structures and modes of chromatography. There has recently been significant growth in the number of such materials in which the base matrix is derivatized to add a covalently attached or grafted polymer layer or, in some cases, a hydrogel that fills the pore space. This review summarizes the main structural and functional features of ion exchangers of this kind, which represent the largest class of such materials. Although the adsorption and transport properties may generally be used operationally and modeled phenomenologically using the same methods as are used for proteins in conventional media, there are noteworthy mechanistic differences in protein behavior in these adsorbents. A fundamental difference in protein retention is that it may be portrayed as partitioning into a three-dimensional polymer phase rather than adsorption at an extended two-dimensional surface, as applies in more conventional media. Beyond this partitioning behavior, however, the polymer-functionalized media often display rapid intraparticle transport that, while qualitatively comparable to that in conventional media, is sufficiently rapid quantitatively under certain conditions that it can lead to clear benefits in key measures of performance such as the dynamic binding capacity. Although possible mechanistic bases for the retention and transport properties are discussed, appreciable areas of uncertainty make detailed mechanistic modeling very challenging, and more detailed experimental characterization is likely to be more productive.

Spiral counter-current chromatography of small molecules, peptides and proteins using the spiral tubing support rotor

An important advance in countercurrent chromatography (CCC) carried out in open flow-tubing coils, rotated in planetary centrifuges, is the new design to spread out the tubing in spirals. More spacing between the tubing was found to significantly increase the stationary phase retention, such that now all types of two-phase solvent systems can be used for liquid–liquid partition chromatography in the J-type planetary centrifuges. A spiral tubing support (STS) frame with circular channels was constructed by laser sintering technology into which FEP tubing was placed in 4 spiral loops per layer from the bottom to the top and a cover affixed allowing the tubing to connect to flow-tubing of the planetary centrifuge. The rotor was mounted and run in a P.C. Inc. type instrument. Examples of compounds of molecular weights ranging from <300 to approximately 15,000 were chromatographed in appropriate two-phase solvent systems to assess the capability for separation and purification. A mixture of small molecules including aspirin was completely separated in hexane–ethyl acetate–methanol–water. Synthetic peptides including a very hydrophobic peptide were each purified to a very high purity level in a sec-butanol solvent system. In the STS rotor high stationary phase retention was possible with the aqueous sec-butanol solvent system at a normal flow rate. Finally, the two-phase aqueous polyethylene glycol–potassium phosphate solvent system was applied to separate a protein from a lysate of an Escherichia coli expression system. These experiments demonstrate the versatility of spiral CCC using the STS rotor.

Weak cation‐exchange monolithic column for capillary liquid chromatography of peptides and proteins

A stable poly(2-carboxyethyl acrylate-co-poly(ethylene glycol) diacrylate) monolith was synthesized inside a 75-μm id capillary by direct in situ photo-initiated polymerization in a binary porogenic solvent consisting of methanol and ethyl ether. The resulting monolith was evaluated for weak cation-exchange capillary liquid chromatography of peptides and proteins. A high dynamic binding capacity of 72.7 mg lysozyme per cm3 column volume was measured. Fast mass transfer was demonstrated by steep breakthrough curves. The resulting monolith exhibited negligible hydrophobicity, leading to good separation of peptides and proteins. Peak capacities of 11 for peptides with a 10-min salt gradient and 39 for proteins with a 20-min salt gradient were measured. An efficiency of 37,000 plates/m for proteins was obtained under isocratic conditions. The effects of functional group concentration, porogenic solvent composition, mobile phase pH, salt gradient rate, and hydrophobicity on the retention of analytes were investigated. Good run-to-run relative standard deviation (RSD) <1.93% and column-to-column RSD <4.63% were achieved.

Intact protein separation by chromatographic and/or electrophoretic techniques for top-down proteomics

Mass spectrometry used in combination with a wide variety of separation methods is the principal methodology for proteomics. In bottom-up approach, proteins are cleaved with a specific proteolytic enzyme, followed by peptide separation and MS identification. In top-down approach intact proteins are introduced into the mass spectrometer. The ions generated by electrospray ionization are then subjected to gas-phase separation, fragmentation, fragment separation, and automated interpretation of mass spectrometric and chromatographic data yielding both the molecular weight of the intact protein and the protein fragmentation pattern. This approach requires high accuracy mass measurement analysers capable of separating the multi-charged isotopic cluster of proteins, such as hybrid ion trap-Fourier transform instruments (LTQ-FTICR, LTQ-Orbitrap). Front-end separation technologies tailored for proteins are of primary importance to implement top-down proteomics. This review intends to provide the state of art of protein chromatographic and electrophoretic separation methods suitable for MS coupling, and to illustrate both monodimensional and multidimensional approaches used for LC-MS top-down proteomics. In addition, some recent progresses in protein chromatography that may provide an alternative to those currently employed are also discussed.

Monolithic capillary columns synthesized from a single phosphate-containing dimethacrylate monomer for cation-exchange chromatography of peptides and proteins

Monoliths containing phosphoric acid functional groups were synthesized from only one monomer, bis[2-(methacryloyloxy)ethyl] phosphate (BMEP), in 75-μm i.d. UV transparent fused-silica capillaries by photo-initiated polymerization for cation exchange chromatography of peptides and proteins. Various synthetic conditions, including porogen solvents, monomer concentration, and polymerization time, were studied. The hydrophobicities of the resulting monoliths were evaluated using propyl paraben under reversed-phase conditions and synthetic peptides under ion-exchange conditions. These monoliths exhibited low hydrophobicities and relatively low porosities due to their highly cross-linked structures. A dynamic binding capacity (lysozyme) of 73 mg/mL of column volume was measured using the best performing monolith. Synthetic peptides were eluted in approximately 30 min without addition of acetonitrile to the mobile phase, yielding a peak capacity of 28. Efficiencies of 52,900 plates/m for peptides and 71,000 plates/m for proteins were obtained under isocratic conditions. The effects of separation conditions, i.e., mobile phase pH and salt gradient rate, were studied. Good run-to-run reproducibility was achieved with a relative standard deviation (RSD) less than 1.5% for retention times of proteins. The column-to-column retention time reproducibility for peptides was less than 3.5% RSD. A monolithic column was used to follow the deamidation of ribonuclease A. The kinetics of deamidation were founded to be first order with a half life of 195 h. A cytochrome C digest was also separated using a linear gradient of sodium chloride.

Hydrophilically Coated Gigaporous Polystyrene Microspheres and their Application in High-Speed Protein Chromatography

Gigaporous poly (styrene-divinylbenzene) (PS) microspheres have been effectively hydrophilized by coating with hydrophobically modified agarose (phenoxyl agarose, Agap). The effect of the phenoxy content on the amount of Agap adsorbed onto the PS surface was examined and various samples of microspheres, differing in surface Agap density, were prepared. After coating, the hydrophilicity of gigaporous PS microspheres was found to be greatly enhanced and the gigaporous character of PS microspheres was well maintained. The modified particles were further coupled with DEAE groups to prepare a DEAE anion exchange medium (DEAE-AP). A column packed with DEAE-AP showed low back pressure and high protein resolution at high flow velocity. Notably, the DEAE-AP column could completely separate a model protein mixture at a flow velocity of 3612 cm/h within 2 min. The results indicate that the gigaporous matrix has considerable advantages for high-speed protein chromatography.

The Cancer/Testis Antigen Prostate-associated Gene 4 (PAGE4) Is a Highly Intrinsically Disordered Protein

The cancer/testis antigens (CTAs) are an important group of heterogeneous proteins that are predominantly expressed in the testis in the normal human adult but are aberrantly expressed in several types of cancers. Prostate-associated gene 4 (PAGE4) is a member of the CT-X family of CTAs that in addition to testis, is highly expressed in the fetal prostate, and may also play an important role both in benign and malignant prostate diseases. However, the function of this gene remains poorly understood. Here, we show that PAGE4 is a highly (100%) intrinsically disordered protein (IDP). The primary protein sequence conforms to the features of a typical IDP sequence and the secondary structure prediction algorithm metaPrDOS strongly supported this prediction. Furthermore, SDS-gel electrophoresis and analytical size exclusion chromatography of the recombinant protein revealed an anomalous behavior characteristic of IDPs. UV circular dichroism (CD) and NMR spectroscopy confirmed that PAGE4 is indeed a highly disordered protein. In further bioinformatic analysis, the PredictNLS algorithm uncovered a potential nuclear localization signal, whereas the algorithm DBS-Pred returned a 99.1% probability that PAGE4 is a DNA-binding protein. Consistent with this prediction, biochemical experiments showed that PAGE4 preferentially binds a GC-rich sequence. Silencing PAGE4 expression induced cell death via apoptosis and in mice carrying PCa xenografts, siRNA-mediated knockdown of the PAGE4 mRNA attenuated tumor growth in vivo. Furthermore, overexpressing PAGE4 protected cells from stress-induced death. To our knowledge, PAGE4 is the first example of a CTA that is an IDP with an anti-apoptotic function.

Development of a displacer-immobilized ligand docking scheme for displacer screening for protein displacement chromatography

Displacement chromatography is a powerful technique for protein purification, but the availability of high-efficacy displacers has greatly limited its applications. In this work, a displacer-immobilized ligand docking scheme was developed for the prediction of displacer efficacy and displacer screening for displacement chromatography of proteins. The structure of immobilized ligand was established by coupling a certain number of ligands to the 3D structure of agarose. A number of known cation, anion and hydrophobic displacers were docked to their respective immobilized ligands to verify the effectiveness of the scheme, and the Spearman ranking correlation coefficients of all cases were over 0.5. The scheme was then used to screen displacers for hydrophobic charge induction chromatography from over 1800 commercially available compounds. Column displacement experiments of several representative compounds showed that the identified displacers were efficacious in the displacement of single component and binary mixtures. It is expected that the combination of the docking scheme with the existing techniques for displacer discovery/design would greatly facilitate the discovery of high-affinity displacers for protein purification.

Polymorphism and pedigree analysis of β-amylase isozymes in Australian wheat

β-Amylase encoded by multiple loci on chromosomes of triticeae crop plants is a starch metabolic enzyme with a significant physiological role in flour functionality. In this study, wheat grain β-amylase isozymes were characterized using an extensive Australian wheat population and genetic breeding/mapping lines. β-amylase isozymes were fractionated by high-resolution native polyacrylamide gel electrophoresis (PAGE) and validated by zymogram and peptide sequencing using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Specific antibodies were generated against the β-amylase purified through protein chromatography and used for β-amylase identification. Further characterization of wheat β-amylases was carried out with immunoblotting and 2-dimensional electrophoresis (2-DE). The enzyme was found to be polymorphic among the Australian and foreign wheat cultivars, with a total of six isozymes: two common and four varying isozymes, the latter group including one novel β-amylase isozyme of fast-mobility in native PAGE, According to pedigree analysis, the origin of this particular isozyme was from the ancestral genotype, Ciano F 67.

Modification of Martini force field for molecular dynamics simulation of hydrophobic charge induction chromatography of lysozyme

Modeling, especially the force field, is crucial for the accuracy of molecular dynamics (MD) simulations. In order for more accurate description of adsorption and desorption behaviors of lysozyme in hydrophobic charge induction chromatography (HCIC), the Martini coarse-grained (CG) force field has been modified based on the statistical analysis and comparison of an all-atom (AA) force field, GROMOS96 43A1, and the Martini force field. The parameters describing the protein–adsorbent interactions have been adjusted to avoid too strong and unrealistic adsorption of lysozyme on the agarose matrix and HCIC ligands. It is found that the adsorption and desorption behaviors monitored using the modified Martini force field and MD simulation are consistent with previous simulation results with 46-bead β-barrel model protein. Repeated adjustment of both protein position and orientation is necessary to generate enough contacts for a stable adsorption. After reducing the pH in the mobile phase, the lysozyme–ligand electrostatic repulsion leads to protein desorption. In the adsorption process, little conformational transition of lysozyme is observed due to its stable structure, which is in line with previous experimental observations. So, it is concluded that after appropriate modification, the Martini force field can be used to examine the HCIC process of lysozyme. The modification strategy has thus extended the applicability of the Martini force field to protein chromatography, and it is expected to facilitate studies of exploring the molecular details in adsorption chromatography of proteins.

Exploiting the interactions between poly-histidine fusion tags and immobilized metal ions

Immobilized metal affinity chromatography (IMAC) of proteins containing poly-histidine fusion tags is an efficient research tool for purifying recombinant proteins from crude cellular feedstocks at laboratory scale. Nevertheless, to achieve successful purification of large amounts of the target protein for critical therapeutic applications that demand the precise removal of fusion tags, it is important to also take into consideration issues such as protein quality, efficiency, cost effectiveness, and optimal affinity tag choice and design. Despite the many considerations described in this article, it is expected that enhanced selectivity, the primary consideration in the field of protein separation, will continue to see the use of IMAC in solving new purification challenges. In addition, the platform nature of this technology makes it an ideal choice in purifying proteins with unknown properties. Finally, the unique interaction between immobilized metal ions and poly-histidine fusion tag has enabled new developments in the areas of biosensor, immunoassay, and other analytical technologies.

Influence of Selected Seminal Plasma Proteins on Mitochondrial Integrity and Speed Parameters of Ram Sperm Stored at Low Temperature

ABSTRACTThe aim of the study was to investigate the effects of separated seminal plasma proteins on the mitochondrial integrity and velocity parameters during low temperature preservation of ram spermatozoa. Gel-filtration chromatography of seminal plasma proteins was performed. Fifteen protein fractions were obtained and SDS-PAGE of separated seminal plasma proteins was run. The results showed that the incubation of spermatozoa at 5°C led to reduction in the number of R123++ dyed cells. The presence of selected proteins with molecular weigh lower than 30 kDa preserved a high number of sperm cells with normal functioning mitochondria (R123++). A positive correlation between the percentage of R123++ reacted sperm cells and the number of progressively motile spermatozoa was registered. The results of computer sperm analyses showed that the proteins with low molecular weight may better protect the spermatozoa velocity parameters—curvilinear velocity, straight-line velocity and average path velocity, than those with higher molecular weight. The use of these selected proteins as biological components to the medium can protect ram spermatozoa and may find practical application in artificial insemination.

Monolithic porous layer open tubular (monoPLOT) columns for low pressure liquid chromatography of proteins.

Glycidyl methacrylate-ethylene dimethacrylate (GMA-co-EDMA) based monolithic porous layer open tubular (monoPLOT) columns (0.05 mm I.D., monolithic layer thickness ≈ 5 µm) have been fabricated using an automated column scanning technique, providing UV polymerisation at 365 nm. Columns were chemically modified to obtain desired diol groups on the surface, and the longitudinal homogeneity of the stationary phase was profiled using scanning capacitively coupled contactless conductivity detector (sC4D), before and after such modification. Using the automated scanning polymerisation technique, column-to-column production reproducibility, including longitudinal phase thickness, was within ±5% RSD. The prepared columns were tested to evaluate their liquid chromatographic stationary phase selectivity, efficiency and reproducibility, with a series of test protein mixtures. Under optimised gradient conditions, the separation of up to 8 proteins was demonstrated on the open tubular column (510 × 0.05 mm I.D.), with a column pressure drop of <1.5 MPa.

Protein Chromatography: Process Development and Scale-Up Giorgio Carta and Alois Jungbauer Wiley-VCH Verlag, Weinheim ISBN 978-3-572-31819-3 346 pages Hard cover

Journal of Separation ScienceVolume 34, Issue 1 p. 118-118 Book Review Protein Chromatography: Process Development and Scale-Up Giorgio Carta and Alois Jungbauer Wiley-VCH Verlag, Weinheim ISBN 978-3-572-31819-3 346 pages Hard cover Frantisek Svec, Frantisek SvecSearch for more papers by this author Frantisek Svec, Frantisek SvecSearch for more papers by this author First published: 20 December 2010 https://doi.org/10.1002/jssc.201090056Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume34, Issue1January 2011Pages 118-118 RelatedInformation

A novel adsorbent for protein chromatography: Supermacroporous monolithic cryogel embedded with Cu2+-attached sporopollenin particles

The aim of this study is to prepare supermacroporous cryogels embedded with Cu2+-attached sporopollenin particles (Cu2+-ASP) having large surface area for high protein adsorption capacity. Supermacroporous poly(2-hydroxyethyl methacrylate) (PHEMA)-based monolithic cryogel column embedded with Cu2+-ASP was prepared by radical cryo-copolymerization of 2-hydroxyethyl methacrylate (HEMA) with N,N′-methylene-bis-acrylamide (MBAAm) as cross-linker directly in a plastic syringe for affinity purification of human serum albumin (HSA). Firstly, Cu2+ ions were attached to sporopollenin particles (SP), then the supermacroporous PHEMA cryogel with embedded Cu2+-ASP was produced by free radical polymerization using N,N,N′,N′-tetramethylene diamine (TEMED) and ammonium persulfate (APS) as initiator/activator pair in an ice bath. Embedded particles (10mg) in PHEMA-based cryogel column were used in the adsorption/desorption of HSA from aqueous solutions. Optimum conditions of adsorption experiments were performed at pH 8.0 phosphate buffer, with flow rate of 0.5mL/min, and at 5°C. The maximum amount of HSA adsorption from aqueous solution was very high (677.4mg/g SP) with initial concentration 6mg/mL. It was observed that HSA could be repeatedly adsorbed and desorbed to the embedded Cu2+-ASP in PHEMA cryogel without significant loss of adsorption capacity.

Hydrophobic interaction chromatography of proteins. IV. Protein adsorption capacity and transport in preparative mode

The adsorption isotherms of four model proteins (lysozyme, α-lactalbumin, ovalbumin, and BSA) on eight commercial phenyl hydrophobic interaction chromatography media were measured. The isotherms were softer than those usually seen in ion-exchange chromatography of proteins, and the static capacities of the media were lower, ranging from 30 to 110mg/mL, depending on the ammonium sulfate concentration and the protein and adsorbent types. The protein-accessible surface area appears to be the main factor determining the binding capacity, and little correlation was seen with the protein affinities of the adsorbents. Breakthrough experiments showed that the dynamic capacities of the adsorbents at 10% breakthrough were 20–80% of the static capacities, depending on adsorbent type. Protein diffusivities in the adsorbents were estimated from batch uptake experiments using the pore diffusion and homogeneous diffusion models. Protein transport was affected by the adsorbent pore structures. Apparent diffusivities were higher at lower salt concentrations and column loadings, suggesting that adsorbed proteins may retard intraparticle protein transport. The diffusivities estimated from the batch uptake experiments were used to predict column breakthrough behavior. Analytical solutions developed for ion-exchange systems were able to provide accurate predictions for lysozyme breakthrough but not for ovalbumin. Impurities in the ovalbumin solutions used for the breakthrough experiments may have affected the ovalbumin uptake and led to the discrepancies between the predictions and the experimental results.

Self-interaction chromatography of proteins on a microfluidic monolith

A novel miniaturized system has been developed for measuring protein–protein interactions in solution with high efficiency and speed, and minimal use of protein. A chromatographic monolith synthesized in a capillary is used in the method to make interaction measurements by self-interaction chromatography (SIC) in a manner that, compared to column methods, is more efficient as well as more readily practicable even if only small amounts of protein are available. The microfluidic monolith requires much less protein for both column preparation and the chromatographic measurements than a conventional SIC system, and in addition offers improved mass transfer and hence higher chromatographic efficiency than for previous SIC miniaturization systems. Protein self-interactions for catalase as a model protein, quantified by measurement of second virial coefficients, B22, were determined by SIC and follow trends that are consistent with previously reported values. Different column derivatization conditions were studied in order to optimize the chromatographic behavior of the microfluidic system for SIC measurements. Chromatographic sensitivity can be further increased by using different column synthesis conditions.