Chromatographic Separation Of Proteins Research Articles

Extractability and chromatographic separation of proteins from potato (Solanum tuberosum L.) trimmings

By-products from the potato processing industry, like potato trimmings, are sustainable sources of proteins. Here, a size-exclusion high performance liquid chromatography (SE-HPLC) method was applied to simultaneously determine the extractability and aggregation state of proteins from three batches of potato trimmings of different cultivars. Obtained SE-HPLC profiles allowed distinguishing between the patatin and protease inhibitor fractions of potato proteins. Moreover, only 75% of the crude proteins could be extracted in phosphate buffer containing sodium dodecyl sulfate and a reducing agent, indicating the presence of physical extraction barriers. Ball milling for 5 min significantly increased protein extractability, but prolonged treatment resulted in aggregation of native patatin and a reduced protein extractability. Microwave-dried trimmings had a lower protein extractability than freeze-dried trimmings. In future research, the SE-HPLC method can be used to examine changes in potato protein (fractions) as a result of processing.

Algorithms to optimize multi-column chromatographic separations of proteins

The goal of this work was to provide a technical solution for the automated optimization of multi-column systems for protein separation and fractionation. Both algorithm and a software that can be downloaded are provided. In this algorithm, the length and order of the individual column segments can be considered. Various solutions are provided by the algorithm, including i) to obtain uniform peak distribution, ii) to park the different species at the inlet of the individual column segments, and iii) to elute all species as a single peak.Two representative examples are presented, showing the possibility to obtain uniform selectivity between monoclonal antibody (mAb) sub-units, and the on-column fractioning of intact mAbs.

Partition Efficiency of Eccentric Coil for Countercurrent Chromatographic Separation of Proteins Using Small-scale Cross-axis Coil Planet Centrifuge with Circular and Elliptic Cylindrical Columns.

The partition efficiency of the double-spaced coil for eccentric and toroidal coils on countercurrent chromatographic separation of proteins was evaluated using the small-scale cross-axis coil planet centrifuge (CPC) equipped with circular and elliptic cylindrical columns. Standard cytochrome c, myoglobin and lysozyme samples were used for separation with the 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate system. In the circular column, the double-spaced eccentric coil yielded better peak resolution than the double-spaced toroidal coil, and the double-spaced eccentric coil yielded better peak resolution than the single-spaced eccentric coil. In the elliptic column, the double-spaced eccentric coil also produced better peak resolution than the double-spaced toroidal coil, but the single-spaced eccentric coil yielded better peak resolution than the double-spaced eccentric coil. The overall results indicated that the double-spaced eccentric coil for the circular column and the single-spaced eccentric coil for the elliptic column yielded better protein separation using the small-scale cross-axis CPC with aqueous two-phase solvent systems.

Separation of monoclonal antibody monomer-dimer mixtures by gradient elution with ceramic hydroxyapatite.

Modeling the chromatographic separations of proteins at manufacturing scale is important since downstream processing costs are often dominant. At such scales, the columns are highly overloaded heightening the challenge of predicting performance. In this work, the separation of a monoclonal antibody monomer-dimer mixture is conducted by gradient elution chromatography with ceramic hydroxyapatite (CHT) columns Type I and Type II under overloaded conditions. Phosphate gradients are shown to be preferable over sodium chloride gradients since the latter result in undesirable pH transitions generated within the column itself. Using sodium phosphate gradients separation is obtained with both CHT types, achieving approximately 90% recovery at 99% monomer purity starting with a mixture containing 30% dimer at total protein loads up to 30 mg/mL. Because of its higher binding capacity, even higher loadings can be obtained with CHT Type I without monomer breakthrough. A hybrid model is developed to describe the separation. The model, based on an empirical description of two-component, competitive isotherms at low sodium phosphate concentration coupled with the stoichiometric displacement model at higher sodium phosphate concentrations, is in good agreement with the experiments using the linear driving force (LDF) approximation to describe adsorption/desorption kinetics. The same LDF rate coefficient predicts the separation at loadings between 0.8 and 30 mg/mL. The model developed in this work can be used as a general tool to optimize operating conditions, understand what factors limit performance, and compare different operating modes.

Purification of Polyhistidine-Tagged Proteins by Immobilized Metal Affinity Chromatography.

Immobilized metal affinity chromatography (IMAC) is based on the affinity of polyhistidine tracts for divalent metal cations (usually Ni2+) immobilized as transition metal chelate complexes on a chromatography resin. The main protocol here is optimized for use of Ni2+-NTA resin to purify soluble 6xHis-tagged proteins by a straightforward batch method during the binding step, followed by gravity flow for washes and elution. This protocol does not require any specialized equipment other than a simple glass or plastic column. IMAC resins can be used in multiple formats, including batch, gravity flow, centrifuge columns, and fast performance liquid chromatography (FPLC) systems. FPLC systems are designed specifically for the chromatographic separations of proteins and other biomolecules. These systems typically contain multiple pumps, an in-line UV absorption monitor, conductivity meter, pH meter, fraction collector, and other options that allow for the simultaneous purification, analysis, and fractionation of the sample. When linked with the appropriate instruments, an FPLC can become a high-precision, automated instrument that separates proteins at a high resolution. An alternative protocol is included here that describes 6xHis-tagged protein purification using FPLC. Procedures for the cleaning and regeneration of the IMAC resin for reuse are also described, and, finally, considerations for storing purified proteins are discussed.

Mechanistic Study of Protein Adsorption on Mesoporous TiO2 in Aqueous Buffer Solutions

Protein adsorption is of fundamental importance for bioseparation engineering applications. In this work, a series of mesoporous TiO2 with various geometric structures and different aqueous buffer solutions were prepared as platforms to investigate the effects of the surface geometry and ionic strength on the protein adsorptive behavior. The surface geometry of the TiO2 was found to play a dominant role in the protein adsorption capacity when the ionic strength of buffer solutions is very low. With the increase in ionic strength, the effect of the geometric structure on the protein adsorption capacity reduced greatly. The change of ionic strength has the highest significant effect on the mesoporous TiO2 with large pore size compared with that with small pore size. The interaction between the protein and TiO2 measured with atomic force microscopy further demonstrated that the adhesion force induced by the surface geometry reduced with the increase in the ionic strength. These findings were used to guide the detection of the retention behavior of protein by high-performance liquid chromatography, providing a step forward toward understanding the protein adsorption for predicting and controlling the chromatographic separation of proteins.

AFM Study of pH‐Dependent Adhesion of Single Protein to TiO2 Surface

AbstractThe effect of pH‐induced electrostatic conditions on the molecular interaction force of a single lysozyme molecule with TiO2 is investigated using atomic force microscopy (AFM). The force between the charged or neutral lysozyme molecule and the TiO2 surface is measured at different pH from 3.6 to 10.8. It is found to be directly proportional to the contact area, given by an effective diameter of the lysozyme molecule, and is further qualitatively verified by the AFM‐measured friction coefficients. The results of the Derjaguin–Landau–Verwey–Overbeek theory show that the pH can change the surface charge densities of both lysozyme and TiO2, but the molecular interaction force at different pH is only dependent on the pH‐induced effective diameter of lysozyme. The molecular interaction forces, quantified at the nanoscale, can be directly used to design high‐performance liquid chromatography measurements at macroscale by tuning the retention time of a protein under varied pH conditions. They can also be applied to develop a model for predicting and controlling the chromatographic separations of proteins.

Fast, low-pressure chromatographic separation of proteins using hydroxyapatite nanoparticles

Columns packed with ultrafine particles (e.g. sub-2 µm porous particles) are suitable for high-resolution, high-speed analytical separation of proteins. However, they require very expensive chromatography systems to provide the ultra-high pressure required for carrying out separations using such columns. Also, frictional heating at high pressure could result in peak broadening and on-column protein degradation. In this paper, we discuss the use of nanoparticles, packed in a box-shaped or cuboid packed-bed device having 50 mm length, 5 mm width, and 3 mm bed-height for fast, high-resolution separation of proteins at low pressure. The low bed height allows the separation to be carried out at low pressure while the cuboid device reduces dispersion effects and thereby keeps the resolution high. Two different types of hydroxyapatite nanoparticles, i.e., needle-shaped (about 20 nm × 150 nm) and spherical (<200 nm) ones, were examined. The experimental results showed that while the needle-shaped nanoparticles were suitable for the separation of small proteins such as lysozyme, the spherical nanoparticles were better suited for separation of larger proteins such as bovine serum albumin and monoclonal antibody. The separation of the two proteins could be carried out in less than 2 min at a pressure lower than 0.8 MPa, using inexpensive chromatography devices a7nd systems, and without high pressure related problems such as frictional heating and on-column protein denaturation.

Characterization of Myoglobin Adsorption into Mesoporous Silica Pores by Differential Scanning Calorimetry.

Adsorption of protein molecules into the pores of a porous material is an important process for chromatographic separation of proteins and synthesis of nanoscale biocatalyst systems; however, there are barriers to developing a method for analyzing the process quantitatively. The purpose of this study is to examine the applicability of differential scanning calorimetry (DSC) for quantitative analysis of protein adsorption into silica mesopores. For this purpose myoglobin, a globular protein (diameter: 35.2 Å) was selected, and its adsorption onto mesoporous silica powders with uniform pore diameters (pore diameters: 39 and 64 Å) was measured by adsorption assay and DSC experiments. Our results confirmed that the adsorption of myoglobin into the silica mesopores induced significant changes in the positions and areas of freezing/melting peaks of the pore water. The decrease in heat of fusion of the pore water after myoglobin adsorption could be utilized to quantify the amount of myoglobin inside the silica mesopores. The advantages of DSC include its applicability to small wet mesoporous silica samples.

Modelling the effect of temperature on the gel-filtration chromatographic protein separation

In this study a mechanistic model for chromatography was taken and the solution to its Laplace transfer function was obtained using the Fast Fourier Transform method. Using previously developed correlations for modelling diffusion, both in solution and intraparticle, and estimating the mass transfer coefficient, the effect of temperature in gel fittration liquid chromatography was investigated. The effect of each individual parameter on the elution curve was systematically explored allowing for reasonable estimates for the different temperature cases. In this case, a system of bovine serum albumin and phenylalanine separated by gel filtration chromatography was simulated to demonstrate how the resolution and the selectivity of the separation will change with physical parameters. Decreasing the particle size and flow rate while increasing the temperature led to higher resolution, which is consistent with experimental literature data.

Effects of pressure and frictional heating on protein separation using monolithic columns in reversed-phase chromatography

Pressure is not typically controlled or adjusted independently of flow rate during method development in reversed-phase LC (RPLC). However, it has been shown that pressure has an effect on analyte molecular molar volume, and the magnitude of this effect is greater for proteins and ionizable compounds than neutral small molecules. This phenomenon has received attention recently in the context of porous sub-2-micron particle packed columns. The present study surveys the effect of pressure and frictional heating on RPLC separations using commercially-available monolithic columns at constant flow rate and with controlled external temperature. Because the current monoliths cannot be operated at high pressures, all experiments were conducted with pressures at or below 200bar. Nonetheless, substantial changes in retention were still observed; for example, an increase in pressure of 75bar shifted the retention factor for bovine insulin from 1.27 to 1.78, a 40% increase, while a similar experiment with the neutral small molecule, toluene, showed no change in retention. Results are presented from investigations of model peptides and proteins ranging in size from 1kDa to 30kDa, as well as experiments performed with a silica-based C18 monolith and a polystyrene divinylbenzene monolith functionalized with a phenyl stationary phase. This work indicates that protein separations in monoliths are highly pressure sensitive, and pressure should therefore be considered as an additional parameter in method development for optimizing retention and selectivity. Given these findings, and the ever-increasing importance of chromatographic separations of proteins in both industrial and academic laboratories, improved instrumentation and mechanisms for directly controlling system backpressure could be of great practical value.

Poly(N-isopropylacrylamide)-grafted dual stimuli-responsive filter paper for protein separation

Thermal and salt dual stimuli-responsive filter-paper-based membranes were prepared by UV-induced grafting of NIPAM-based polymers on paper surface. The grafting ratio could be controlled by monomer concentration during grafting polymerization. The results from pressure drop measurement of the mobile phase flowed cross the membrane demonstrate that an appropriate grafting ratio would be 8%–10%. Protein adsorption on the membrane through hydrophobic interaction could be promoted by increasing temperature and lyotropic salt concentration. The effect of grafted polymer structure on protein binding performance was studied. Filter paper grafted with NIPAM-based branched copolymer consisting of hydrophobic monomer moieties shows ten times higher protein binding capacity than that of the original filter paper. The separation of plasma proteins using the dual stimuli-responsive membrane was examined to demonstrate feasible application for hydrophobic interaction chromatographic separation of proteins.

Countercurrent chromatographic separation of proteins using an eccentric coiled column with synchronous and nonsynchronous type-J planetary motions.

Protein separation was performed using the high-speed countercurrent chromatograph (HSCCC) at both synchronous and nonsynchronous type-J planetary motions. The partition efficiency was evaluated with two different column configurations, eccentric coil and toroidal coil, on the separation of a set of stable protein samples including cytochrome C, myoglobin and lysozyme with a polymer phase system composed of 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate. Better peak resolution was obtained by the eccentric coil than by the toroidal coil using either lower or upper phase as the mobile phase. The peak resolution was further improved using the eccentric coil by the nonsynchronous type-J planetary motion with the combination of 1066 rpm of column rotation and 1000 rpm of revolution.

Springer Protocols and Wiley Current Protocols

Perhaps the most valuable resource in a research lab is the battered, chemically stained, and much used binder chock full of protocols. Protocols are practical methods that describe the design and implementation of an experiment. Rather than reinventing the wheel, protocols allow scientists to take an existing scaffold and tweak it for their specific experimental needs, saving time and effort. Most importantly, protocols help set a standardized method of research, which ultimately fosters scientific reproducibility. Good protocols can also provide details on how to use complex instrumentation, how to create buffers and other chemical mixtures to exacting standards, and how to analyze and interpret data. They are an important cornerstone to successful scientific experimentation. For years, in addition to the highly customized lab notebooks maintained by individual researchers, scientists have referenced the “gold standard” peer-reviewed, and updated print protocols found in Current Protocols in Molecular Biology and Methods in Molecular Biology. Both feature protocols on topics both general and highly specific, along with additional information including comprehensive subject overviews. Unlike the methods sections in most papers, the protocols featured in Current Protocols and Methods in Molecular Biology are rich with information, including discussion on theory, practical tips, buffer recipes, and troubleshooting advice. Additionally, the protocols are oftentimes linked into useful thematic groupings, providing an excellent mechanism for discovery of alternate approaches if one experimental avenue is unsuccessful. Current Protocols was purchased by Wiley and has now expanded to seventeen titles that are electronically available. These include the original Current Protocols in Molecular Biology but now cover other areas such as neuroscience, bioinformatics, and genetics. Likewise, Methods in Molecular Biology has been optioned by Springer and now contains a family of fifteen electronic titles, including biochemistry, infectious disease, and cell biology. These electronic databases are designed for any scientist looking for background information in detailed protocols on a particular technique, although graduate students will benefit the most from the detailed, value-added sections such as “history,” “commentary,” and “troubleshooting.” Advanced scientists will be unlikely to use these resources as they will have likely developed a strong list of protocols tailored to their specific research projects. Major features of the online content of Current Protocols include 15,000 procedures and techniques, extensive background information, and thematically grouped protocols that facilitate discovery of alternative experimental approaches. A new tools section provides 19 different tools to perform routine calculations such as buffer pH or molecular weight. The tools section also includes databases of reference information such as the CD Antigen database. Interestingly, an ImageJ applet is available for image manipulation. However, the value-added tools section is not easy to navigate to from a specific title (such as “Essential Laboratory Techniques”) and requires users to find the Current Protocols main page. Springer Protocols contain 18,000 protocols, many with extensive supplemental information in addition to the core protocols. Springer Protocols also includes an online community, which gives registered researchers the opportunity to upload their own protocols and to leave comments on other protocols. Although this feature is intriguing, researchers are unlikely to store personal protocols in their Springer accounts, and very few protocols have any comments. The primary utility of the Springer account lies in mobile access: users can login and receive all the content to which their institutions subscribe on their mobile devices in an easy-to-read format. Both Current Protocols and Springer Protocols are regularly updated by professionals in the field. Libraries have the option of licensing one or all of the individual titles. The biggest barrier to use lies in the classification of the database: Both Springer Protocols and Current Protocols are thought of as either a journal or as a book by scientists. A scientist trying to find a protocol on the chromatographic separation of proteins by looking under “Current Protocols in Molecular Biology” as a journal rather than as an electronic monograph could get quite exasperated. Even more frustratingly, Springer Protocols are often cataloged under individual subtitles, which can make discovery difficult. Way-finding guides for both Springer and Current Protocols would greatly facilitate use for scientists but would need to be created at each institution individually to address the specific details of how the resources are cataloged. Additionally, as the specificity of the protocol subtitles increases, institutions are presented with the challenge of sometimes only offering some of the titles, which can lead to confusion regarding accessibility. However, despite these hazards of classification, both Current Protocols and Springer Protocols are extremely useful electronic resources that are rich with content and utility.

Temperature-responsive membrane for hydrophobic interaction based chromatographic separation of proteins in bind-and-elute mode

Butyl-acrylate (BA) containing branched poly-(N-isopropyl acrylamide) or PNIPAM copolymer was grafted on filter paper to prepare composite membrane for carrying out temperature-responsive hydrophobic interaction membrane chromatography (HIMC) of proteins. The copolymer demonstrated two-step phase transition behavior due to the presence of BA and PNIPAM, making the changeover from hydrophilic to hydrophobic state more tunable. The feasibility of using the temperature-responsive membrane was demonstrated by controlling human immunoglobulin G (hIgG) binding and release using temperature change. Higher temperature promoted protein binding and lower temperature favored protein release in this process, which was carried out using lyotropic salt-free mobile phase. The purification of monoclonal antibody (mAb) hIgG1-CD4 from simulated CHO cell culture supernatant was carried out to demonstrate practical application for the novel temperature-responsive membrane.

High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study

The retention and elution of proteins in ion-exchange chromatography is routinely controlled by adjusting the mobile phase salt concentration. It has repeatedly been observed, as judged from adsorption isotherms, that the apparent heterogeneity of adsorption is lower at more-eluting, higher ionic strength. Here, we present an investigation into the mechanism of this phenomenon using a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT). We observed that the number of functional adsorption sites was smaller at high ionic strength and that these sites had reduced desorption kinetic heterogeneity, and thus narrower predicted elution profiles, for the anion-exchange adsorption of α-lactalbumin on an agarose-supported, clustered-charge ligand stationary phase. Explanations for the narrowing of the functional population such as inter-protein interactions and protein or support structural changes were investigated through kinetic analysis, circular dichroism spectroscopy, and microscopy of agarose microbeads, respectively. The results suggest the reduction of heterogeneity is due to both electrostatic screening between the protein and ligand and tuning the steric availability within the agarose support. Overall, we have shown that single molecule spectroscopy can aid in understanding the influence of ionic strength on the population of functional adsorbent sites participating in the ion-exchange chromatographic separation of proteins.

Molecular Theory of Protein Sorption on Weak Polyelectrolyte Gel-Modified Charged Surface

We developed a detailed molecular theory to determine protein adsorption on a charged surface as a function of the pH, salt concentration, protein concentration and surface charge density, as well as the modification of the surface by the attachment of a weak polyelectrolyte gel film. The approach includes specific molecular details of the proteins, their translational and rotational degrees of freedom as well as the acid-base equilibrium of the titratable amino acids. We also include the conformational degrees of freedom and acid-base equilibrium of the polymeric gel, the solution entropic contributions, and electrostatic and excluded-volume interactions. The experimental titration curve of lysozyme and the isoelectric point (IP) in solution are reproduced successfully. In low salt conditions, lysozyme adsorption profiles as a function of pH on a non-modified surface are broad and have a maximum value at a pH lower than the isoelectric point of the protein. When the salt concentration is increased, maximum adsorption occurs at evenlower pH values. The level of protein adsorption on a gel-modified surface is two orders of magnitude greater than on a non-modified surface, and has a narrower maximum close to the IP at low salt concentration. The molecular theory may explain the excellent properties of such modified surfaces for chromatographic separation of proteins as well as help to design new chromatographic systems. Moreover, the theory provides with a fundamental understanding of the competition between the different contribution that determine the adsorption behavior.

Evaluating the performance of different multicolumn setups for chromatographic separation of proteins on hydrophobic interaction chromatography media by a numerical study

A theoretical study has been performed on the effectiveness of isolating a target component out of a multi-component protein mixture using different arrangements of chromatographic columns. Three continuous systems have been considered which were able to perform solvent gradient separations, such as: open loop simulated moving bed, countercurrent solvent gradient purification and carousel multicolumn setup. The performance of the continuous processes was examined with respect to productivity, yield and eluent consumption and compared to a single-column batch system. As a case study separation of a ternary mixture of proteins on HIC media has been selected. Two separation problems have been analyzed referring to the situation when the target component was the most strongly adsorbed as well as when it exhibited intermediate adsorption strength. A mathematical model has been used to simulate the process dynamics and to optimize operating conditions for the separation. The numerical study indicated that batch column arrangements can outperform SMB-based configurations regarding all performance indicators considered, which has been attributed to solvent mixing in the recycled streams and distortion of the gradient shape in SMB units. It has been concluded that the performance of complex multicolumn systems should be verified vs. batch column operations prior to the realization of the separation process.

Preparation of weak cation exchange packings for chromatographic separation of proteins using ``click chemistry''

"Click chemistry" is defined as a class of robust and selective chemical reactions affording high yields and is tolerant to a variety of solvents (including water), functional groups, and air. In this study, click chemistry was used as an effective strategy for coupling three alkyne-carboxylic acids onto the azide-silica to obtain three novel stationary phases of weak cation exchange chromatography, which were characterized with FTIR and elemental analysis. Six kinds of standard proteins, such as myoglobin, RNase A, RNase B, cytochrome C, α-chymotrypsin A, and lysozyme, were separated completely with the three novel weak cation exchange chromatography stationary phases. Compared with commercial weak cation exchange chromatography columns, the three kinds of novel weak cation exchange chromatography packings prepared by click chemistry approach have better resolution and selectivity. The mass recovery of more than 97% was obtained for all the tested proteins, and the bioactivity recovery of lysozyme on the prepared column was determined to be 96%. In addition, lysozyme was purified successfully from egg white with the novel weak cation exchange chromatography column by one step. The purity was more than 97% and a high specific activity was achieved to be 81 435 U/mg. The results illustrate the potential of click chemistry for preparing stationary phase for ion-exchange chromatography.

High-density lipoprotein loses its anti-inflammatory capacity by accumulation of pro-inflammatory-serum amyloid A

High-density lipoprotein (HDL) is known to have potent anti-inflammatory properties. Monocyte chemoattractant protein-1 is an important pro-inflammatory cytokine in early atherogenesis. There is evidence that HDL can lose its protective function during inflammatory disease. In patients with end-stage renal disease (ESRD), epidemiological studies have documented that the inverse correlation between HDL-cholesterol and cardiovascular risk is lost. Many structural modifications leading to reduced HDL function have been characterized, but the functional consequences are not fully understood. We showed that HDL from patients with ESRD has a lower anti-inflammatory potential by reduced inhibition of monocyte chemoattractant protein-1 formation in vascular smooth muscle cells. Via a proteomic approach, we identified proteins in HDL from ESRD patients exerting pro-inflammatory actions. By chromatographic separation of proteins and mass-spectrometric analysis, we found serum amyloid A (SAA) to be one molecule acting as a potent pro-inflammatory protein. SAA is enriched in HDL from ESRD patients, correlating with reduced anti-inflammatory capacity. In SAA signal transduction, activation of formyl-peptide receptor 2 is involved. SAA enrichment in HDL of healthy subjects reduced the anti-inflammatory capacity of HDL and correlated with its decreased function. These results suggest that SAA enrichment of HDL during disease conditions contributes to the decreased protective function. It is a novel finding that SAA acts as a pro-inflammatory molecule to reduce the anti-inflammatory properties of HDL.