Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports.

A poly-His tag was fused in the glutaryl acylase (GA) from Acinetobacter sp. strain YS114 cloned in E. coli yielding a fully active enzyme. Biochemical analyses showed that the tag did not alter the maturation of the chimeric GA (poly-His GA) that undergoes a complex post-translational processing from an inactive monomeric precursor to the active heterodimeric enzyme. This enzyme has been used as a model to develop a novel and very simple procedure for one-step purification of poly-His proteins via immobilized metal-ion affinity chromatography on tailor-made supports. It was intended to improve the selectivity of adsorption of the target protein on tailor-made chelate supports instead of performing a selective desorption. The rate and extent of the adsorption of proteins from a crude extract from E. coli and of pure poly-His tagged GA on different metal chelate supports was studied. Up to 90% of proteins from E. coli were adsorbed on commercial chelate supports having a high density of ligands attached to the support through long spacer arms, while this adsorption becomes almost negligible when using low ligand densities, short spacer arms and Zn2+ or Co2+ as cations. On the contrary, poly-His GA adsorbs strongly enough on all supports. A strong affinity interaction between the poly-His tail and a single chelate moiety seems to be the responsible for the adsorption of poly-His GA. By contrast, multipoint weak interactions involving a number of chelate moieties seem to be mainly responsible for adsorption of natural proteins. By using tailor-made affinity supports, a very simple procedure for one-step purification of GA with minimal adsorption of host proteins could be performed. Up to 20 mg of GA were adsorbed on each ml of chelate support while most of accompanying proteins were hardly adsorbed on such supports. Following few washing steps, the target enzyme was finally recovered (80% yield) by elution with 50 mM imidazole with a very high increment of specific activity (up to a 120 purification factor).     

Affinity chromatography of polyhistidine tagged enzymes. New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions

New immobilized metal ion affinity chromatography (IMAC) matrices containing a high concentration of metal-chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the purification of polyhistidine (poly-His) tagged proteins. The purification of an interesting recombinant multimeric enzyme (a thermoresistant beta-galactosidase from Thermus sp. strain T2) has been used to check the performance of these new chromatographic media. IMAC supports with a high concentration (and surface density) of metal chelate groups promote a rapid adsorption of poly-His tagged proteins during IMAC. However, these supports also favor the promotion of undesirable multi-punctual adsorptions and problems may arise for the simple and effective purification of poly-His tagged proteins: (a) more than 30% of the natural proteins contained in crude extracts from E. coli become adsorbed, in addition to our target recombinant protein, on these IMAC supports via multipoint weak adsorptions; (b) the multimeric poly-His tagged enzyme may become adsorbed via several poly-His tags belonging to different subunits. In this way, desorption of the pure enzyme from the support may become quite difficult (e.g., it is not fully desorbed from the support even using 200 mM of imidazole). The coating of these IMAC supports with dextrans greatly reduces these undesired multi-point adsorptions: (i) less than 2% of natural proteins contained in crude extracts are now adsorbed on these novel supports; and (ii) the target multimeric enzyme may be fully desorbed from the support using 60 mM imidazole. In spite of this dramatic reduction of multi-point interactions, this dextran coating hardly affects the rate of the one-point adsorption of poly-His tagged proteins (80% of the rate of adsorption compared to uncoated supports). Therefore, this dextran coating of chromatographic matrices seems to allow the formation of strong one-point adsorptions that involve small areas of the protein and support surface. However, the dextran coating seems to have dramatic effects for the prevention of weak or strong multipoint interactions that should involve a high geometrical congruence between the enzyme and the support surface.     

Selective extraction of low molecular weight proteins by mesoporous silica particles with modified internal and external surfaces

A new mesoporous silica material with modified external and internal surfaces (alkyl diol–vinyl-SiO₂) was prepared and applied to selectively extract low molecular weight (LMW) proteins and peptides (less than 11 kDa) from biological samples. X-ray diffraction, N₂ adsorption, transmission electron microscopy, and Fourier transform infrared spectroscopy were used to characterize the alkyl diol–vinyl-SiO₂. This material was used to extract LMW proteins (peptides) from standard proteins, a mixture of peptides, and crude human plasma. We compared the amounts of different proteins adsorbed by alkyl diol–vinyl-SiO₂ and by vinyl-SiO₂ (vinyl group on the internal surface of the mesoporous silica material). The desorption recovery of alkyl diol–vinyl-SiO₂ by different eluents was also investigated by using a standard protein (insulin) as a model LMW protein. The material could efficiently extract LMW proteins and peptides from a mixture of standard proteins and crude human plasma with good extraction efficiency and desorption recovery. Furthermore, the alkyl diol–vinyl-SiO₂ had much better extraction selectivity for LMW proteins and peptides than a commercial C₁₈ solid phase extraction material.     

Frontal analysis for characterizing the adsorption-desorption behavior of beta-lactoglobulin on immunoadsorbents

High-performance frontal affinity chromatography was employed to study the adsorption-desorption kinetics characterizing the retention of beta-lactoglobulin (beta-LG) onto polyclonal anti-beta-lactoglobulin (anti-beta-LG) chromatographic supports. The adsorption and desorption processes were studied by analyzing two different elution fronts separated by a relatively long rinsing step. The method consists in performing two successive frontal injections of the protein. In between, the column was rinsed with a given volume of mobile phase (buffer alone). During this rinsing stage, a partial desorption may occur and a novel amount of protein could be adsorbed in the second frontal injection step. The whole process (first adsorption, possible desorption, and second adsorption) was simulated by a numerical procedure, in which the column was divided into a large number of slices. A model based on bi-Langmuir type kinetics was used to describe the adsorption of the protein on the support. The model assumes a non-uniform adsorbent with two types of binding sites. At equilibrium the adsorption isotherm is of the bi-Langmuir type. A global adsorption effect was considered which includes the effective binding process and the mass transfer resistances due to the transport to the binding site. Therefore, the column capacity and the kinetic parameters of the model (apparent adsorption and desorption rate constants) were determined from the best fit of the first and second adsorption fronts to the experimental ones. The other parameters of the model are the saturation capacities for the adsorption on each type of sites. The equilibrium affinity constants were estimated in a single experiment from the ratio of the apparent adsorption and desorption rate constants. The high values found (around 10(8) M(-1)) reveal a strong interaction of beta-LG with the immunoadsorbent. Kinetic measurements were carried out at different flow rates. Both the apparent adsorption and desorption kinetics were faster at larger flow rates, indicating an important contribution of the mass transfer resistance in the stagnant fluid at the particle boundary. However, as expected, close values were found for the resulting equilibrium constants calculated from the ratio of the apparent adsorption and desorption rate constant determined at various flow rates.     

Diagnostic protein discovery using proteolytic peptide targeting and identification

Plasma protein profiling with mass spectrometry is currently being evaluated as a diagnostic tool for cancer and other diseases. These experiments consist of three steps: plasma protein fractionation, analysis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), and comparisons of the MALDI profiles to develop diagnostic fingerprints using bioinformatic techniques. While preliminary results appear promising in small sample groups, the method is limited by the sensitivity of MALDI-MS for intact proteins, the limited mass range of MALDI-MS, and difficulties associated with isolating individual proteins for identification to validate the diagnostic fingerprint. Here we present an alternative and improved method directed toward diagnostic protein discovery, which incorporates proteolytic peptide profiling, bioinformatic targeting of ion signals, and MALDI tandem mass spectrometry (MS/MS) peptide sequencing, rather than fingerprinting. Pancreatic cancer patients, pancreatitis patients, and controls are used as the model system. Profiling peptides after enzymatic digestion improves sensitivity and extends the accessible protein molecular weight range when compared to intact protein profiling. The first step is to extract and fractionate the proteins from plasma. Each fraction is digested with trypsin and subsequently analyzed by MALDI-MS. Rather than using bioinformatic analysis as a pattern-matching technique, peptides are targeted based on the disease to control peak intensity ratios measured in the averages of all mass spectra in each group and t-tests of the intensity of each individual peak. The targeted peptide ion signals are subsequently identified using MALDI-MS/MS in quadrupole-TOF and tandem-TOF instruments. This study found not only the proteins targeted and identified by a previous protein profiling experiment, but also detected additional proteins. These initial results are consistent with the known biology of pancreatic cancer or pancreatitis, but are not specific to those diseases.     

Improved selection of LMW over HMW proteins from human plasma by mesoporous silica particles with external modification

Selective extraction of low molecular weight (LMW) proteins and peptides from complex biological samples plays an important role in the discovery of useful biomarkers and signaling molecules. Various methods, such as solid-phase extraction (SPE), ultrafiltration, and size-exclusion chromatography have been developed for such extraction purpose. In this study, we present, to our knowledge, the first demonstration of alkyl-diol@SiO₂ mesoporous material MCM-41 (alkyl-diol group on the external surface of mesoporous material) for selective extraction of LMW proteins and peptides from complex biological samples. The adsorption kinetics of LMW proteins, the influence of pH on adsorption and the desorption recovery by different elution solvents were investigated by using standard proteins as model samples. It was demonstrated that the modification of alkyl-diol group on the external surface could efficiently decrease the adsorption of HMW protein and increase the desorption recovery of LMW protein. Furthermore, the mesoporous materials were applied to selectively extract LMW proteins and peptides (<10 kDa) from crude human plasma. And the modified MCM-41 material had much better extraction selectivity and efficiency for LMW proteins and peptides than unmodified one.     

Characterizing the adsorption of proteins on glass capillary surfaces using electrospray-differential mobility analysis

We quantify the adsorption and desorption of a monoclonal immunoglobulin-G antibody, rituxamab (RmAb), on silica capillary surfaces using electrospray-differential mobility analysis (ES-DMA). We first develop a theory to calculate coverages and desorption rate constants from the ES-DMA data for proteins adsorbing on glass capillaries used to electrospray protein solutions. This model is then used to study the adsorption of RmAb on a bare silica capillary surface. A concentration-independent coverage of ≈4.0 mg/m(2) is found for RmAb concentrations ranging from 0.01 to 0.1 mg/mL. A study of RmAb adsorption to bare silica as a function of pH shows maximum adsorption at its isoelectric point (pI of pH 8.5) consistent with literature. The desorption rate constants are determined to be ≈10(-5) s(-1), consistent with previously reported values, thus suggesting that shear forces in the capillary may not have a considerable effect on desorption. We anticipate that this study will allow ES-DMA to be used as a "label-free" tool to study adsorption of oligomeric and multicomponent protein systems onto fused silica as well as other surface modifications.     

Large entropy difference between terrace and step sites on surfaces

Using atomic beam/surface scattering measurements to investigate the desorption kinetics of low-coverage Pb from Mo(100), we uncover a large entropy difference between Pb atoms at terrace and step sites, which should be general for adsorbates on surfaces at high temperatures. A line shape analysis of the transient desorption signal reveals the presence of two species with different lifetimes on the surface. An Arrhenius analysis of these lifetimes from 1150 to 1320 K provides the prefactors and desorption activation energies (332 and 411 kJ/mol) of these two states. A comparison of these energies to those measured directly via adsorption calorimetry strongly suggests that one state is a terrace-bound species. The other, more strongly bound species is attributed to steps. The more strongly bound step species has the higher rate constant for desorption because of its much larger desorption prefactor (9 x 10 (19) vs 5 x 10 (15) s (-1)). Within transition state theory, the ratio of these prefactors corresponds to 82 J/(mol K) higher entropy for the terrace species than for the step species. This large entropy difference is quantitatively reproduced by a simple model which assumes the terrace species is a 2D ideal gas parallel to the surface and the step species is a 1D ideal gas along the step edges. Such a difference will generally exist for adsorbed species when k B T exceeds the barrier height for adsorbate diffusion across terraces. A consequence of this large entropy difference is that the defect sites are much less populated relative to terrace sites than would be expected based on enthalpy alone. The measured prefactor for Pb desorption was used to analyze earlier surface lifetime measurements for Pb on MgO(100) to extract adsorption energies for that system, as well.     

Electrostatic model for protein adsorption in ion-exchange chromatography and application to monoclonal antibodies,lysozyme and chymotrypsinogen A

A model for the adsorption equilibrium of proteins in ion-exchange chromatography explicitly accounting for the effect of pH and salt concentration in the limit of highly diluted systems was developed. It is based on the use of DLVO theory to estimate the electrostatic interactions between the charged surface of the ion-exchanger and the proteins. The corresponding charge distributions were evaluated as a function of pH and salt concentration using a molecular approach. The model was verified for the adsorption equilibrium of lysozyme, chymotrypsinogen A and four industrial monoclonal antibodies on two strong cation-exchangers. The adsorption equilibrium constants of these proteins were determined experimentally at various pH values and salt concentrations and the model was fitted with a good agreement using three adjustable parameters for each protein in the whole range of experimental conditions. Despite the simplifications of the model regarding the geometry of the protein–ion-exchanger system, the physical meaning of the parameters was retained.     

Incorporating water-release and lateral protein interactions in modeling equilibrium adsorption for ion-exchange chromatography

The equilibrium adsorption of two albumin proteins on a commercial ion exchanger has been studied using a colloidal model. The model accounts for electrostatic and van der Waals forces between proteins and the ion exchanger surface, the energy of interaction between adsorbed proteins, and the contribution of entropy from water-release accompanying protein adsorption. Protein-surface interactions were calculated using methods previously reported in the literature. Lateral interactions between adsorbed proteins were experimentally measured with microcalorimetry. Water-release was estimated by applying the preferential interaction approach to chromatographic retention data. The adsorption of ovalbumin and bovine serum albumin on an anion exchanger at solution pH>pI of protein was measured. The experimental isotherms have been modeled from the linear region to saturation, and the influence of three modulating alkali chlorides on capacity has been evaluated. The heat of adsorption is endothermic for all cases studied, despite the fact that the net charge on the protein is opposite that of the adsorbing surface. Strong repulsive forces between adsorbed proteins underlie the endothermic heat of adsorption, and these forces intensify with protein loading. It was found that the driving force for adsorption is the entropy increase due to the release of water from the protein and adsorbent surfaces. It is shown that the colloidal model predicts protein adsorption capacity in both the linear and non-linear isotherm regions, and can account for the effects of modulating salt.     

高比表面季铵基树脂的结构调控及在三七叶总皂甙纯化中的应用研究

利用Davankov后交联反应,合成了一类兼具高比表面积和高功能基含量的季铵基(—N+(CH)3)吸附树脂,考察了树脂比表面积和功能基含量的调控规律,并将其用于三七叶总皂甙的进一步纯化.结果表明,当树脂比表面积为692m2/g,交换量为2.1mmol/g时,树脂具有最佳的纯化效果,只通过吸附—解吸一步工艺,产品纯度即可从32.0%提高到90%以上,皂甙的回收率高于95%.最后,初步探讨了树脂对皂甙和色素的吸附机理,认为树脂对皂甙的吸附是单纯的疏水性作用力,而对色素的吸附应为疏水-离子交换双重作用的协同效应.     

A comprehensive study of concepts and phenomena of the nonspecific adsorption of beta-lactoglobulin.

We investigate nonspecific protein adsorption processes by comparing experimentally measured adsorption kinetics of beta-lactoglobulin with mathematical models. The adsorption and desorption behavior of this protein on a hydrophilic glass surface in citrate buffer (pH 3.0), monitored for a large set of different bulk concentrations (0.5x10(-8) M-1.5x10(-6) M) using a supercritical angle fluorescence (SAF) biosensor, is reported. Increasing adsorption rates and overshootings in the beginning of the adsorption are observed as well as a transition to an almost irreversibly bound state of the protein in the long term. Furthermore, rinsing experiments prove that adsorbed proteins abruptly change their desorption behavior from irreversible to reversible when a critical surface coverage theta(crit) is reached. Based on all experimental observations, a mathematical model composed of three adsorbed states differing in their surface affinity is proposed. Terms to account for lateral interactions between surface-bound proteins are included, which yield an excellent fit of the measured kinetics. For the first time, several phenomena that have been discussed in theoretical studies are confirmed by comparing experimental data with a single model.     

Molecularly Imprinted Supermacroporous Cryogels for Myoglobin Recognition

Myoglobin is a primary iron, and oxygen-binding protein of muscle tissues and levels can be an important diagnostic biomarker for acute myocardial infarction, myocardial necrosis, or other cardiac diseases. The establishment of myoglobin recognition systems is important because of its protein’s structural and functional values in physiology, biochemistry, and diagnostic value in some damaged muscle tissue and cardiac diseases. For this purpose, we used molecular imprinting technique for myoglobin recognition from aqueous solutions and human plasma. In the first step, myoglobin-imprinted poly(hydroxyethyl methacrylate) (PHEMA) cryogels (MGb-MIP) were prepared, and optimum myoglobin adsorption conditions were determined. Selectivity experiments have been done with the competitive proteins, and myoglobin adsorption from IgG and albumin-free human plasma was studied. The purity of the desorbed samples was determined with SDS-PAGE. The desorption efficiency and reusability of the MGb-MIP cryogels were tested, and it was shown that without any significant loss in the adsorption capacity, MGb-MIP cryogels can be used a number of times for myoglobin recognition and separation.     

Enzyme capturing and concentration with mixed matrix membrane adsorbers

This study reports the use of membrane adsorbers for lysozyme (LZ) capturing and concentration: the membrane adsorbers are prepared by incorporation of ion exchange resins into an EVAL porous matrix. The mixed matrix membrane (MMM) adsorber possesses an open and interconnected porous structure with a large ion exchange surface available for enzyme adsorption. The adsorptive membrane features both a high static as well as a high dynamic LZ adsorption capacity. The measured LZ adsorption isotherm is of the Langmuir type, with a maximum adsorption capacity of 147 mg LZ/ml membrane. Dynamic LZ adsorption capacity at a flux of 25 l/h/m² was 63 mg LZ/ml membrane, which is significantly higher than the equivalent commercial membrane Sartobind C. Since the kinetics of desorption processes are faster than the kinetics of adsorption processes, the performance can be improved by exerting the desorption processes at higher fluxes than the adsorption processes. The MMM can be reused in multiple adsorption/desorption cycles maintaining the high binding capacity performance. Fluorescence spectra of the LZ after adsorption and elution were similar to native LZ. This is confirmed by activity tests showing that the activity of LZ was maintained after an adsorption and desorption cycle.     

A simple and efficient protocol for large‐scale preparation of three flavonoids from the flower of Daphne genkwa by combination of macroporous resin and counter‐current chromatography

In this paper, macroporous resin column chromatography and counter‐current chromatography (CCC) were applied for large‐scale preparative separation of three flavonoids from the flower of Daphne genkwa, a famous Chinese medicinal herb. Nine kinds of resins were investigated by adsorption and desorption tests and D101 macroporous resin was selected for the first cleaning‐up, in which 40% aqueous ethanol was used to remove the undesired constituents and 90% aqueous ethanol was used to elute the targets. The crude extract after the first step was directly subjected to the preparative CCC purification using the solvent system composed of n‐hexane–ethyl acetate–methanol–water (4:5:4:5, v/v). The compounds apigemin (823 mg), 3‐hydroxyl‐genkwanin (842 mg) and genkwanin (998 mg) with the purities of 98.79, 97.71 and 93.53%, respectively, determined by HPLC were produced from 3‐g crude extract only in one CCC run. Their chemical structures were identified by MS, UV and the standards.     

The application of macroporous resins in the separation of licorice flavonoids and glycyrrhizic acid

Glycyrrhizic acid (GA) and licorice flavonoids (LF) are the two classes of bioactive components in licorice with known pharmacological effects. But long-term excessive intake of GA may cause sodium retention and hypertension. In this study, the performance and adsorption characteristics of four widely used macroporous resins for the separation of deglycyrrhizinated, flavonoids enriched licorice has been critically evaluated. The sorption and desorption properties of LF and GA on macroporous resins including XDA-1, LSA-10, D101 and LSA-20 have been compared. The adsorption capacity was found to depend strongly on the pH of the feed solution. XDA-1 offers much higher adsorption capacity for GA and LF than other resins, and its adsorption data fit the best to the Freundlich isotherm. XDA-1 also shows much higher adsorption affinity towards LF than that of GA based on calculated results from the measured adsorption isotherms. Dynamic adsorption and desorption experiments have been carried out on a XDA-1 resin packed column to obtain optimal parameters for separating GA and LF. An enriched LF extract (about 21.9% purity) free of GA, and an enriched GA extract with 66% purity can be separated from crude licorice extract in one run.     

Adsorption kinetics of protein mixtures. A tentative explanation of “the vroman effect”

A model for protein adsorption kinetics is presented. This model includes diffusion limited adsorption, adsorption and desorption rate constants which are dependent on the surface concentration and an interaction term for the mutual influence of the adsorbed protein molecules. It is shown that, in first approximation, the values of the adsorption and desorption rate constants are exponential functions of the surface concentration. Assuming an adequate interaction term it is possible to show with this model for the adsorption kinetics of a mixture of proteins that the ratio of the adsorbed proteins is strongly dependent on the overall surface concentration even if the ratio of the bulk concentrations of these proteins is kept constant. Differences in interaction terms for the different proteins offer a possible explanation for the peculiar behaviour of plasma protein adsorption on a surface at different dilutions of the plasma, the so called “Vroman effect”.     

The role of ligands on protein retention in adsorption chromatography: A surface energetics approach

Protein adsorption onto hydrophobic chromatographic supports has been investigated using a colloid theory surface energetics approach. The surface properties of commercially available chromatographic beads, Toyopearl Phenyl 650‐C, and Toyopearl Butyl 650‐C, have been experimentally determined by contact angle and zeta potential measurements. The adsorption characteristics of these beads, which bear the same backbone matrix but harbor different ligands, have been studied toward selected model proteins, in the hydrated as well as dehydrated state. There were two prominent groups of proteins observed with respect to the chromatographic supports presented in this work: loosely retained proteins, which were expected to have lower average interaction energies, and the strongly retained proteins, which were expected to have higher average interaction energies. Results were also compared and contrasted with calculations derived from adsorbent surface energies determined by inverse liquid chromatography. These results showed a good qualitative agreement, and the interaction energy minima obtained from these extended Derjaguin, Landau, Verwey and Overbeek calculations were shown to correlate well with the experimentally determined adsorption behavior of each protein.     

A green and efficient protocol for large‐scale production of glycyrrhizic acid from licorice roots by combination of polyamide and macroporous resin adsorbent chromatography

A green and efficient method for large‐scale preparation of glycyrrhizic acid from licorice roots was developed by combination of polyamide and macroporous resin. The entire preparation procedure consisted of two simple separation steps. The first step is to use polyamide resin to remove licorice flavoniods from the licorice crude extract. Subsequently, various macroporous resins were tried to purify glycyrrhizic acid, and HPD‐400 showed the most suitable adsorption and desorption properties. Under the optimized conditions, a large‐scale preparation of glycyrrhizic acid from licorice roots was carried out. A 20 kg raw material produced 0.43 kg of glycyrrhizic acid using green aqueous ethanol as the solvent. The purity of glycyrrhizic acid was increased from 11.40 to 88.95% with a recovery of 76.53%. The proposed method may be also extended to produce large‐scale other triterpenoid saponins from herbal materials.     

Electrochemical quartz crystal nanobalance study of the adsorption/displacement phenomena of proteins and lipids on Pt

The electrochemical quartz crystal nanobalance (EQCN) was used to measure the adsorption behavior of a series of lipids (stearate, oleate, linoleate, and gamma-linolenate) on a Pt surface from a phosphate buffer pH 7.0 solution at 295 K and to investigate their adsorption/displacement behavior with the proteins, beta-lactoglobulin and alpha-lactalbumin, which are known to cause fouling during milk processing. The EQCN technique and the complementary technique of cyclic voltammetry measured simultaneously provided information on the efficiency of solubilization of the proteins by these lipids. Excellent agreement was obtained for the surface concentration of adsorbed lipid from the surface charge density from cyclic voltammetry measurements and the change in mass from the EQCN frequency measurements. The Gibbs energy of adsorption showed the lipids to have a strong affinity for the platinum surface. Addition of protein to a preadsorbed lipid layer showed alpha-lactalbumin to be able to coadsorb with the lipids, while beta-lactoglobulin was able to desorb some of the unsaturated lipids but appeared to coadsorb with the saturated lipid, stearate. Addition of lipid to a preadsorbed protein layer showed the unsaturated lipids to be able to displace some of the protein. A comparison of the desorption ability of the lipids showed stearate to be very inefficient at removing protein, while the other three lipids were able to remove each of the proteins, with the order of efficiency for protein desorption being oleate > linoleate > gamma-linolenate.