Biomedical subjects

Xiao-Yan Dong

Publications and source records attributed to Xiao-Yan Dong.

13 recordsLinked to original sources

PubMed2006

Oscillatory transverse electric field enhances protein resolution and capacity of size-exclusion chromatography.

Protein separations by a novel size-exclusion electrochromatography (SEEC) are presented. The present SEEC, denoted as pSEEC, was established with an oscillatory low-voltage electric field perpendicular to the mobile-phase streamline. Retention experiments with different proteins indicated that the influence of electric field strength on the partition coefficient is different for different proteins as well as for the same protein under different mobile-phase conditions. These results of protein retention led to the experimental design of protein separations with binary mixtures of BSA and immunoglobulin G (IgG), myoglobin (Myo) and lysozyme (Lys), as well as ovalbumin (Oval) and Myo. The separation results for the binary protein systems sufficiently exhibited the applicability of the pSEEC for various separations in terms of their molecular weights (MWs) as well as pIs. For example, it was possible to separate the gel-excluded proteins (BSA/IgG) as well as gel-permeable and similar-molecular-weight proteins (Myo/Lys) by the pSEEC. Moreover, in the cases of Oval/ Myo, which could be partially separated by size-exclusion chromatography, the use of the pSEEC greatly improved the resolution and the separation became possible at high sample loading. The results indicate that the pSEEC technology is promising for preparative protein separations.

Animals↗

PubMed2006

High-capacity purification of hen egg-white proteins by ion-exchange electrochromatography with an oscillatory transverse electric field.

The ion-exchange electrochromatography with an oscillatory electric field perpendicular to the mobile-phase flow driven by pressure (pIEEC) was used to separate hen egg-white (HEW) proteins. The results were compared with those of normal ion-exchange chromatography (IEC). The column was designed as three-compartment rectangular column of 2-mL with dimensions (length x width x depth) of 40 x 10 x 5 mm(3) and the electric field was applied across the direction of column width. Q Sepharose FF was packed into the central compartment as the chromatographic bed. It was confirmed that the dynamic binding capacity (DBC) of different proteins (ovotransferrin and ovalbumin) in the HEW solution increased 2.3 times when an oscillatory electric current of 30 mA at 1/20 Hz was applied in the transverse column direction. Then, the HEW proteins were separated by the pIEEC at loading amounts 2.3-fold higher than those by the IEC. When the feedstock of about one-third of the DBC was applied to the columns (i.e., 7 mL for the pIEEC and 3 mL for the IEC), similar separation efficiencies of the two chromatographic modes were achieved. Both the recovery yield and purity reached 73% to over 90%. The results indicate that the pIEEC is promising for high-capacity purification of proteins.

Animals↗

PubMed2005

Effect of hexanol on the reversed micelles of Span 85 modified with Cibacron Blue F-3GA for protein solubilization.

Sorbitan trioleate (Span 85) modified with Cibacron Blue F-3GA (CB) was used as an affinity surfactant (CB-Span 85) to form affinity-based reversed micelles in n-hexane. It was found that the addition of hexanol to the reversed micellar system resulted in a significant increase in water content and hydrodynamic radius of the affinity-based reversed micelles. Moreover, the reversed micelles with hexanol revealed broader aggregation number distribution and larger average aggregation number than the reversed micelles without hexanol addition. This is considered to be due to the decreases in the micellar curvature and rigidity of the micellar interfacial layer and the increase in the micellar interfacial fluidity. Consequently, the solubilization capacity of lysozyme increased about 70% in the reversed micellar solution with 3 vol% hexanol. On the other hand, the capacity of BSA was only 30% increased under the same conditions due to its larger molecular size than lysozyme. Kinetic analysis revealed that the increase in the micellar interfacial fluidity in the presence of hexanol resulted in faster release of lysozyme from the micelles, thus leading to an increase of the overall volumetric mass transfer coefficient in the back extraction.

Hexanols↗

PubMed2005

Characterization of reversed micelles of Cibacron Blue F-3GA modified Span 85 for protein solubilization.

The nonionic surfactant of sorbitan trioleate (Span 85) was modified with Cibacron Blue F-3GA (CB) as an affinity surfactant (CB-Span 85) to form affinity-based reversed micelles in n-hexane. The reversed micelles formed by the mixture of Span 85 and CB-Span 85 conjugate were extensively characterized in water content, hydrodynamic radius, and aggregation number. The results show that the water content and hydrodynamic radius of the reversed micelles were significantly increased by the introduction of CB ligands (CB-Span 85 conjugate), and the reversed micelles with CB-Span 85 conjugate had a wider aggregation number distribution than the Span 85 reversed micelles. Using lysozyme as a model protein, protein solubilization by the reversed micelles was investigated. Lysozyme solubilization increased significantly with the coupled CB concentration, indicating that the extraction was based upon the affinity interactions between lysozyme molecules and the CB ligand. High solubilization of lysozyme was obtained by the affinity-based reversed micelles of 62.7 mmol/L Span 85 with coupled CB higher than 0.25 mmol/L. Lysozyme recovery was carried out using a stripping solution of high ionic strength. The recovered lysozyme exhibited an activity equivalent to the native lysozyme and its secondary structure was also unchanged. The results indicate that the reversed micellar system would find potential application in protein separation.

Hexanes↗

PubMed2004

Refolding kinetics of denatured-reduced lysozyme in the presence of folding aids.

The refolding kinetic behavior of denatured-reduced lysozyme in the presence of folding aids (acetamide, acetone, thiourea, L-arginine or glycerol) was studied utilizing a simplified model describing the competition between first-order folding reaction and third-order aggregation. It was found that the protein folding aids could be categorized into two groups. One of them at proper concentrations, such as acetamide, acetone, thiourea and L-arginine, stabilized unfolded protein or folding intermediates. In the presence of these additives, the folding rate decreased with increasing their concentration, and there existed a concentration where the aggregation rate constant was minimized. So, there was an optimum concentration for the folding aids to produce a high yield. The other group was protein stabilizers such as glycerol. In the presence of this kind of folding aids, both the refolding rate and yield were enhanced by increasing their concentration to a proper value. Moreover, their effect on improving protein refolding was additive to those of the first group. So the cooperative application of the two kinds of folding aids could result in favorable refolding rate and yield of protein.

Complex Mixtures↗

PubMed2003

Modeling of the whole expanded-bed protein adsorption process with yeast cell suspensions as feedstock.

Expanded bed adsorption of bovine serum albumin (BSA) directly from a feedstock containing whole yeast cells has been investigated with an anion-exchanger DEAE Spherodex M. In the presence of 6% (w/w) yeast cells, the axial liquid-phase dispersion coefficient was found in the order of 10(-6) m2/s, which felled into the common range of 1.0 x 10(-6)-1.0 x 10(-5) m2/s observed previously without the use of cell suspensions as mobile phase. We found that the static and dynamic binding capacity of BSA decreased with increasing the yeast cell concentration due to the competitive adsorption of cells onto the outer surface of the anion-exchanger. However, because of the small size of the adsorbent, the large pore diffusivity of protein and the favorable column efficiency (low axial dispersion coefficient), the dynamic binding capacity of BSA in the presence of 6% (w/w) cells in the expanded bed reached 86% that of the equilibrium adsorption density. Then, the whole expanded bed adsorption process of BSA in the presence of cells, including feedstock loading, washing and elution steps, was predicted using a mathematical model with parameters all determined independently. In the elution stage, the steric mass-action adsorption isotherm with salt concentration as one of the model parameters was used to predict the step-gradient elution process with salt concentration increases. Computer simulations showed that the model was in good agreement with the experimental results for the whole operation process.

Adsorption↗

PubMed2002

Analysis of diffusion models for protein adsorption to porous anion-exchange adsorbent.

The ion-exchange adsorption kinetics of bovine serum albumin (BSA) and gamma-globulin to an anion exchanger, DEAE Spherodex M, has been studied by batch adsorption experiments. Various diffusion models, that is, pore diffusion, surface diffusion, homogeneous diffusion and parallel diffusion models, are analyzed for their suitabilities to depict the adsorption kinetics. Protein diffusivities are estimated by matching the models with the experimental data. The dependence of the diffusivities on initial protein concentration is observed and discussed. The adsorption isotherm of BSA is nearly rectangular, so there is little surface diffusion. As a result, the surface and homogeneous diffusion models do not fit to the kinetic data of BSA adsorption. The adsorption isotherm of gamma-globulin is less favorable, and the surface diffusion contributes greatly to the mass transport. Consequently, both the surface and homogeneous diffusion models fit to the kinetic data of gamma-globulin well. The adsorption kinetics of BSA and gamma-globulin can be very well fitted by parallel diffusion model, because the model reflects correctly the intraparticle mass transfer mechanism. In addition, for both the favorably bound proteins, the pore diffusion model fits the adsorption kinetics reasonably well. The results here indicate that the pore diffusion model can be used as a good approximate to depict protein adsorption kinetics for protein adsorption systems from rectangular to linear isotherms.

Adsorption↗

PubMed2002

[hEPO transfer and expression by a recombinant adeno-associated virus vector].

In order to achieve sustained human erythropoietin (hEPO) expression in vivo for the treatment of anemias, recombinant adeno-associated virus (rAAV)-mediated hEPO transfer was studied in this report. rAAV vector plasmid carrying hEPO was constructed, and rAAV vector cell line for production of rAAV was also established. By using the one helper virus-one vector cell line strategy, which we reported previously, rAAV containing the hEPO expression cassette was produced in large-scale. The results showed that the hEPO expressed effectively by rAAV-mediated hEPO transfer into cultured BHK-21 cells. Intramuscular injection of rAAV-hEPO to Balb/c mice resulted in the in vivo expression of hEPO for at least ten weeks, along with the significant elevation of the hematocrits. This report indicates the potential use of rAAV-mediated gene transfer for the treatment of various anemias.

Animals↗

PubMed2001

Effect of Guanidinium Chloride Concentration on the Renaturation of Denatured Lysozyme.

The effect of guanidinium chloride (GdmCl) on the renaturation of denatured-reduced chicken egg white lysozyme was studied. It was found that the renaturation yield was significantly related to the GdmCl concentration, that is, the GdmCl concentration required to obtain a high renaturation yield increased with the increase of the denatured lysozyme concentration. When denatured lysozyme concentrations were lower than 0.21 g/L, the presence of 0.7 mol/L GdmCl resulted in the complete renaturation of lysozyme, while higher GdmCl concentration, i.e., 1.0--1.5 mol/L, was needed to yield more than 95% renaturation of 0.6--1.05 g/L of the denatured lysozyme. In addition, lysozyme renaturation rate decreased with increasing GdmCl concentration. Therefore, it is considered that optimizing GdmCl concentration is the key step to enhance the renaturation yield of denatured proteins.

Journal Article↗

PubMedDate not supplied

Cooperative effect of artificial chaperones and guanidinium chloride on lysozyme renaturation at high concentrations.

It has been recognized that the artificial chaperone system, cetyltrimethylammonium bromide and beta-cyclodextrin, is effective for enhancing protein renaturation. In this work, we studied the effect of the artificial chaperone system and guanidinium chloride (GdmCl) on the oxidative renaturation of lysozyme at 0.21-1.05 mg/mL, and a kinetic model based on the competition between protein folding and aggregation was employed to express the renaturation process. The refolding rate constant increased, while the aggregation rate constant decreased, with increasing concentration of the artificial chaperones. With increasing GdmCl concentration (0.28-2 M), both rate constants decreased, but there existed a specific GdmCl concentration that maximized the ratio of the two rate constants and thus the renaturation yield. The results obviously indicated the cooperative effect of GdmCl and the artificial chaperones on enhancing protein renaturation.

Guanidine↗

PubMedDate not supplied

Expanded bed adsorption of protein with DEAE Spherodex M.

In this article, the bed expansion behavior and the hydrodynamic and protein adsorption properties of the DEAE Spherodex M in expanded bed with mobile phases of different viscosities have been studied. The axial liquid-phase dispersion coefficient is found to be on the order of 10(-6) m(2)/s, falling into the common range from 1.0 x 10(-6) to 1.0 x 10(-5) m(2)/s observed previously in expanded bed operation. Because of the small size of the adsorbent, the high pore diffusivity of protein and the favorable column efficiency (low dispersion coefficient), the dynamic binding capacity (DBC) of bovine serum albumin (BSA) at 5% breakthrough in the expanded bed reaches over 80% that of the equilibrium adsorption density (EAD). Moreover, a theoretical model with unadjustable model parameters is used for the prediction of the breakthrough curves. Computer simulations show that the model agrees well with the experimental results at breakthrough less than about 50%. It indicates that the model is promising in the prediction of protein breakthrough behavior because breakthrough profiles at 5-50% breakthrough points are more important in practical applications.

Adsorption↗

PubMedDate not supplied

Modeling and simulation of fed-batch protein refolding process.

The simplified kinetic model that assumes competition between first-order folding and third-order aggregation was used to model the fed-batch refolding of denatured-reduced lysozyme. It was found that the model was able to describe the process at limited concentration ranges, i.e., 1-2 and 5-7 mg mL(-)(1), respectively, at extensive guanidinium chloride (GdmCl) concentrations and controlled concentrations of oxidizing and reducing agents. The folding or aggregation rate constant was different at the two protein concentration ranges and strongly dependent on the denaturant concentration. As a result, both rate constants at the two concentration ranges were expressed as functions of GdmCl concentration. The rate constants determined by fed-batch experiments could be employed for the prediction of the fed-batch process but were not able to be extended to a batch refolding by direct dilution. Computer simulations show that the denaturant concentration and fed-batch flow rate are important factors influencing the refolding yield. Prolonged fed-batch time is beneficial to keep the transient intermediate concentration at a low level and to increase the yield of correctly folded protein. This is of importance when the denaturant concentration in refolding buffer solution is low. Thus, at a low denaturant concentration, fed-batch time should be sufficiently long, whereas at an appropriately high GdmCl concentration, a short fed-batch time or a high feed rate of the denatured protein is effective to give a high refolding yield.

Guanidine↗

PubMedDate not supplied

Protein refolding mediated by reverse micelles of Cibacron Blue F-3GA modified nonionic surfactant.

An affinity-based reverse micellar system formulated with nonionic surfactant was applied to the refolding of denatured-reduced lysozyme. The nonionic surfactant of sorbitan trioleate (Span 85) was modified with Cibacron Blue F-3GA (CB) as an affinity surfactant (CB-Span 85) to form affinity-based reverse micelles in n-hexane. The water content of 15 was found optimal for lysozyme refolding in the reverse micellar system of 62.7 mmol/L Span 85 with coupled CB of 0.3 and 0.5 mmol/L. In addition, the operating conditions such as pH and the concentrations of urea and redox reagents were optimized. Under the optimized conditions, complete renaturation of lysozyme at 3-3.5 mg/mL was achieved, whereas dilution refolding in the bulk aqueous phase under the same conditions gave much lower activity recovery. Moreover, the secondary structure of the refolded lysozyme was found to be the same as the native lysozyme. Over 95% of the refolded lysozyme was recovered from CB-Span 85 reverse micelles by a stripping solution of 0.5 mol/L MgCl(2). Thus, the present system is advantageous over the conventional reverse micellar system formed with ionic surfactants in the ease of protein recovery.

Animals↗