Oxidative refolding of the denatured/reduced lysozyme was investigated by using weak-cation exchange chromatography (WCX). The stationary phase of WCX binds to the reduced lysozyme and prevented it from forming inter...Oxidative refolding of the denatured/reduced lysozyme was investigated by using weak-cation exchange chromatography (WCX). The stationary phase of WCX binds to the reduced lysozyme and prevented it from forming intermolecular aggregates. At the same time urea and ammonium sulfate were added to the mobile phase to increase the elution strength for lysozyme. Ammonium sulfate can more stabilize the native protein than a common eluting agent, sodium chloride. Refolding of lysozyme by using this WCX is successfully. It was simply carried out to obtain a completely and correctly refolding of the denatured lysozyme at high concentration of 20.0 mg/mL.展开更多
The renaturation and purification of recombinant human proinsulin (rh-proinsulin) expressed in E. coli with the unit of simultaneous renaturation and purification of protein (USRPP) in semi-preparative scale was studi...The renaturation and purification of recombinant human proinsulin (rh-proinsulin) expressed in E. coli with the unit of simultaneous renaturation and purification of protein (USRPP) in semi-preparative scale was studied. The result shows that rh-proinsulin extracted with 8.0 mol/L urea can be renatured and purified simultaneously in 45 minutes with the USRPP (1050 mm ID). The purity of rh-proinsulin was found to be more than 90% and the mass recovery to be more than 80%. The renaturation effect of rh-proinsulin with the USRPP was tested by enzyme cleavage for obtaining insulin. In addition, the result was further confirmed with RPLC, SDS-PAGE electrophoresis, and MALDI-TOF, respectively.展开更多
The refolding of the reduced/denatured insulin from bovine pancreas as the model protein was investigated with weak anion exchange chromatography (WAX) coupled with MALDI-TOF MS. The results indicated that the disul...The refolding of the reduced/denatured insulin from bovine pancreas as the model protein was investigated with weak anion exchange chromatography (WAX) coupled with MALDI-TOF MS. The results indicated that the disulfide bonds almost cannot be formed correctly with the common mobile phase by WAX. However, with the urea gradient elution and in the presence of GSSG/ Cyst as the ratio 1:6 in the mobile phase employed, the disulfide exchange of reduced/denatured insulin can be accelerated resulting in forming the correct three disulfide bonds. The protein refolding efficiency of reduced/denatured insulin can be increased from 3 % to 34%. The effects of urea gradient and the oxidant and reductant groups, such as GSSG/GSH, Cyst, and GSSG/Cyst, on the forming the disulfide bonds of reduced/denatured insulin were investigated in detail. The results were further tested by the separation of the WAX fraction of reduced/denatured insulin with RPLC and MALDI-TOF MS.展开更多
The thermal behaviors of five proteins in hydrophobic interaction chromatography (HIC) were investigated in the temperature range from 0 to 50℃. The thermodynamic parameters (△H°,△S°, △Cp°and △G...The thermal behaviors of five proteins in hydrophobic interaction chromatography (HIC) were investigated in the temperature range from 0 to 50℃. The thermodynamic parameters (△H°,△S°, △Cp°and △G°) of these proteins in the process of retention and unfolding were determined. The existence of enthalpy and entropy convergence with temperature was confirmed. The differences of the isoentropic and isoenthalpic temperatures for protein unfolding in HIC system from the traditional solution were elucidated.展开更多
A new method with only a single chromatographic column to accomplish the separation of intact protein molecules by the combination of ion exchange chromatography and hydrophobic interaction chromatography is firstly p...A new method with only a single chromatographic column to accomplish the separation of intact protein molecules by the combination of ion exchange chromatography and hydrophobic interaction chromatography is firstly presented. By selecting twice a suitable stationary phase, mobile phase, buffer exchange condition, and sample injection, some proteins usually required to separate with two-dimensional chromatography can now be accomplished only using the single column in one hour. In addition, the separated proteins can maintain their original three-or four-dimensional molecular structure. It would be expected that this method can also provide a new thought for sample pre-frac-tionation in proteomic investigation, especially, for the limitation of sample source.展开更多
Substantial attention has been paid to the stoi-chiometric displacement models for solute retention (SDM-R) in liquid chromatography and adsorption (SDM-A) at surfaces since they were developed. The SDM has a strongly...Substantial attention has been paid to the stoi-chiometric displacement models for solute retention (SDM-R) in liquid chromatography and adsorption (SDM-A) at surfaces since they were developed. The SDM has a strongly theoretical basis and has evolved to the point that it is widely applied in chemistry, biochemistry, molecular biology and biotechnology. This review introduces the history, recent developments, and new concepts relating to the SDM, including theory, mathematical expressions, and applications across a broad range of fields.展开更多
Artificial molecular chaperone (AMC) and ion exchange chromatography (IEC) were integrated, thus a new refolding method, artificial molecular chaperone-ion exchange chromatography (AMC-IEC) was developed. Compar...Artificial molecular chaperone (AMC) and ion exchange chromatography (IEC) were integrated, thus a new refolding method, artificial molecular chaperone-ion exchange chromatography (AMC-IEC) was developed. Compared with AMC and IEC, the activity recovery of lysozyme obtained by AMC-IEC was much higher in the investigated range of initial protein concentrations, and the results show that AMC-IEC is very efficient for protein refolding at high concentrations. When the initial concentration of lysozyme is 180 mg/mL, its activity recovery obtained by AMC-IEC is still as high as 76.6%, while the activity recoveries obtained by AMC and IEC are 45.6% and 42.4%, respectively.展开更多
The monodisperse poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads with macroporous in the range of 8.0—12.0 m were prepared by a single-step swelling and polymerization method. The seed particles prepared...The monodisperse poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads with macroporous in the range of 8.0—12.0 m were prepared by a single-step swelling and polymerization method. The seed particles prepared by dispersion polymerization exhibited good absorption of the monomer phase. The pore size distribution of the beads was evaluated by gel permeation chromatography and mercury intrusion method. By using this media, a weak cation exchange (WCX) stationary phase for HPLC was synthesized by a new chemical modification method. The prepared resin has advantages of biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery and good resolution for proteins. The measured bioactivity recovery for lysozyme was (96±5)%. The dynamic protein loading capacity of the synthesized WCX packings was 21.3 mg/g. Five proteins were completely separated in 8.0 min using the synthesized WCX stationary phase. The experimental results show that the obtained WCX resin has very weak hydrophobicity. The WCX resin was also used for the rapid separation and purification of lysozyme from egg white in 8 min with only one step . The purity and specific bioactivity of the purified lysozyme was found more than 92.0% and 70184 U/mg, respectively.展开更多
In this work, a kind of preparation method of zwitterionic ion chromatography (ZIC) stationary phase modified with phosphorylcholine (PC) was obtained by hydrolyzing after bonding phosphorylcholine dichloride to d...In this work, a kind of preparation method of zwitterionic ion chromatography (ZIC) stationary phase modified with phosphorylcholine (PC) was obtained by hydrolyzing after bonding phosphorylcholine dichloride to diol-silica to better explore the characteristics of the PC groups as ZIC stationary phase ligand in simultaneous separation of acidic proteins and basic proteins. The results showed that tv^o kinds of acidic proteins and three kinds of basic proteins can be separated completely, meanwhile, hen egg white was separated and purified and three kinds of egg white components ovalbumin, G2 ovoglobulin and ovotransfemin proteins were separated completely by one single step on PC-ZIC column, the purity of all proteins reached above 95%. PC-ZIC stationary phase was successfully improved with better separation capacity and selectivity than previously reported in this paper.展开更多
A new approach for separation of intact proteins with high resolution, high speed and high sample loading ("three highs") by non-porous packings in high performance liquid chromatography is presented.A chrom...A new approach for separation of intact proteins with high resolution, high speed and high sample loading ("three highs") by non-porous packings in high performance liquid chromatography is presented.A chromatographic cake having larger diameter than its thickness is firstly employed to completely separate 1.0 and 40 μg of the mixture of 7 standard proteins within 1 min un-der conventional chromatographic conditions.Also, 0.5 mg of the mixture was almost completely separated within 1 min, accom-plishing the "three high" purpose.This research expands the application utilized for separation of intact proteins with non-porous packings.A smaller geometric size of chromatographic cake packed with much smaller particles of non-porous packings may improve results.展开更多
Minor-adjustment of the retention of peptides, induced by varying the mobile phase flow-rate(MPF-R), is a new dynamic separation method for simultaneously and rapidly identifying and improving the selectivity of hidde...Minor-adjustment of the retention of peptides, induced by varying the mobile phase flow-rate(MPF-R), is a new dynamic separation method for simultaneously and rapidly identifying and improving the selectivity of hidden and overlapping peptide peaks. It can also-stabilize the reverse elution order of some pair-peaks under gradient elution in reverse phase liquid chromatography. The retention characteristics of peptides under gradient elution in RPLC was firstly found to be dominated by two variables of the steady region(SR) and migration region(MR). The changes in peptide retention induced by varying the MPF-R can be attributed to changes in the rate of bond breaking of multiple molecular interactions of peptides from the SR and of the mass transfer of peptides from the stationary phase to the mobile phase in the MR. The two dynamic variables were also found to independently depend on the type of peptide. Desirable results were obtained using six standard oligopeptides and a real sample of trypsin-digested lysozyme.It is expected that the quality control of peptide drugs, high dispersion of peptide peaks in peptide mapping and "bottom-up MS"in proteomics will be improved by this method, even enabling peptide purification on a preparative scale in industry.展开更多
The addition of packing material for high performance hydrophobic interaction chromatograghy (HPHIC) into the denaturant solution to prevent, or depress protein aggregation in the denatuaration process is presented....The addition of packing material for high performance hydrophobic interaction chromatograghy (HPHIC) into the denaturant solution to prevent, or depress protein aggregation in the denatuaration process is presented. The renaturation of α-chymotrypsin (α-Chy) denatured with guanidine hydrochloride (GuHCl) solution indicated that renaturation efficiency can be enhanced from 36.1% to 59.0% by this new method. The structure of the ligand linking of HPHIC packings is also important for the protein renaturation.展开更多
Based on the monodisperse poly(glycidyl methacrylate-co-ethylenedimethacrylate) beads (PGMA/EDMA) with macropore as a medium, a new hydrophilic medium cation exchange (MCX) stationary phase for HPLC was synthesi...Based on the monodisperse poly(glycidyl methacrylate-co-ethylenedimethacrylate) beads (PGMA/EDMA) with macropore as a medium, a new hydrophilic medium cation exchange (MCX) stationary phase for HPLC was synthesized by a new chemically modified method. The stationary phase was evaluated with the property of ion exchange, separability, reproducibility, hydrophilicity, effect of salt concentration, salt types, column loading and pH on the separation and retention of proteins in detail. It was found that it follows ion exchange chromatographic (IEC) retention mechanism. The measured bioactivity recovery for lysozyme was (96 ± 5)%. The dynamic protein loading capacity of the synthesized MCX packings was 21.8 mg/g. Five proteins were almost completely separated within 6.0 min at a flow rate of 4 mL/min using the synthesized MCX resin. The MCX resin was also used for the rapid separation and purification of lysozyme from egg white with only one step. The purity and specific bioactivity of the purified lysozyme was found more than 95% and 70345 U/mg, respectively.展开更多
Ionic liquids(ILs) immobilized on silica as novel high performance liquid chromatography(HPLC)stationary phases have attracted considerable attention. However, it has not been applied to protein separation. In thi...Ionic liquids(ILs) immobilized on silica as novel high performance liquid chromatography(HPLC)stationary phases have attracted considerable attention. However, it has not been applied to protein separation. In this paper, N-methylimidazolium IL-modified silica-based stationary phase(Silpr Mim)was prepared and investigated as a novel multi-interaction stationary phase charged positively for protein separation. The results indicate that all of the basic proteins tested cannot be absorbed on this novel stationary phase, whereas all of the acidic proteins tested can be retained, and the baseline separation of eight kinds of acidic protein standards can be achieved when performed in reversed phase/ion-exchange chromatography(RPLC/IEC) mode. Compared with commonly used commercial octadecylated silica(ODS) column, the novel stationary phase can show selectivity and good resolution to acidic proteins, which has a promising application in the separation and analyses of acidic proteins from the complex samples in proteomics. In addition, the chromatographic behavior of proteins, the effect of the ligand structure and the retention mechanism on this stationary phase were also investigated.展开更多
The renaturation of the denatured α-chymotrypsin (α-Chy) with 1.7 mol · L-1 guanidine hydrochloride (GuHCI) by three kinds of stationary phase of high performance hydrophobic interaction chromatography (STHIC) ...The renaturation of the denatured α-chymotrypsin (α-Chy) with 1.7 mol · L-1 guanidine hydrochloride (GuHCI) by three kinds of stationary phase of high performance hydrophobic interaction chromatography (STHIC) with a comparable hydrophobicity but different ligand structures was investigated. The obtained result indicates that the ligand structures of the three STHIC contribute to the renaturation efficiency of α-Chy in the order of the end ligands PEG-600< phenyl group < tetrahydrofurfuryl alcohol (THFA).展开更多
In previous paper a new adsorption isotherm which relates the amount of solute absorbed to the solvent concentration is proposed and simplified, and it only can be used at lower solute concentration. In this article t...In previous paper a new adsorption isotherm which relates the amount of solute absorbed to the solvent concentration is proposed and simplified, and it only can be used at lower solute concentration. In this article the scope of the new adsorption isotherm is extended and the expressions with three and four parameters are obtained. The equations with multi parameters are valid when the adsorbed amounts are larger and show non linear logarithmic relationships. Tests with a homologue of aromatic alcohols by frontal analysis in reversed phase liquid chromatography demonstrate that the experimental results fit those equations well. In addition, the predicted values by the multi parameters were found to fit the experimental values well also. The parameters have physical meaning only for the two parameter equation for the aromatic alcohols.展开更多
The refolding of the reduced-denatured insulin from bovine pancreas was investigated with the size exclusion chromatography (SEC). It was shown that the reduced-denatured insulin originally denatured with 7.0 mol·...The refolding of the reduced-denatured insulin from bovine pancreas was investigated with the size exclusion chromatography (SEC). It was shown that the reduced-denatured insulin originally denatured with 7.0 mol·L-1 guanidine hydrochloride (GuHCl) or 8.0 mol·L-1 urea could not be refolded with a non-oxidized mobile phase. Although the oxidized and reduced glutathione (GSSG and GSH) were employed in the oxidized mobile phase, the reduced-denatured insulin still could not be renatured. However, in the presence of 2.0 mol·L-1 urea in the oxidized mobile phase employed, the reduced-denatured insulin can be refolded with SEC, and the aggregation of denatured insulin can be diminished by urea. In addition, the disulfide exchange of reduced-denatured insulin also can be accelerated with GSSG/GSH in the oxidized mobile phase. The three disulfide bridges of insulin were formed correctly and the reduced-unfolded insulin can be renatured completely. The results were further tested with reversed-phase liquid chromatography (RPLC) and hydrophobic interaction chromatography (HIC).展开更多
With frontal analysis (FA), the dependence of adsorption isotherms of insulin on the composition of mobile phase in reversed phase liquid chromatography (RPLC) has been investigated. This is also a good example to em...With frontal analysis (FA), the dependence of adsorption isotherms of insulin on the composition of mobile phase in reversed phase liquid chromatography (RPLC) has been investigated. This is also a good example to employ the stoichiometric displacement theory (SDT) for investigating solute adsorption in physical chemistry. Six kinds of mobile phase in RPLC were employed to study the effects on the elution curves and adsorption isotherms of insulin. The key points of this paper are: (1) The stability of insulin due to delay time after preparing, the organic solvent concentration, the kind and the concentration of ion pairing agent in mobile phase were found to affect both elution curve and adsorption isotherm very seriously. (2) To obtain a valid and comparable result, the composition of the mobile phase employed in FA must be as same as possible to that in usual RPLC of either analytical scale or preparative purpose. (3) Langmuir Equation and the SDT were employed to imitate these obtained adsorption isotherms. The expression for solute adsorption from solution of the SDT was found to have a better elucidation to the insulin adsorption from mobile phase in RPLC.展开更多
基金This work is supported by the National Natural Science Foundation(No.20175016).
文摘Oxidative refolding of the denatured/reduced lysozyme was investigated by using weak-cation exchange chromatography (WCX). The stationary phase of WCX binds to the reduced lysozyme and prevented it from forming intermolecular aggregates. At the same time urea and ammonium sulfate were added to the mobile phase to increase the elution strength for lysozyme. Ammonium sulfate can more stabilize the native protein than a common eluting agent, sodium chloride. Refolding of lysozyme by using this WCX is successfully. It was simply carried out to obtain a completely and correctly refolding of the denatured lysozyme at high concentration of 20.0 mg/mL.
文摘The renaturation and purification of recombinant human proinsulin (rh-proinsulin) expressed in E. coli with the unit of simultaneous renaturation and purification of protein (USRPP) in semi-preparative scale was studied. The result shows that rh-proinsulin extracted with 8.0 mol/L urea can be renatured and purified simultaneously in 45 minutes with the USRPP (1050 mm ID). The purity of rh-proinsulin was found to be more than 90% and the mass recovery to be more than 80%. The renaturation effect of rh-proinsulin with the USRPP was tested by enzyme cleavage for obtaining insulin. In addition, the result was further confirmed with RPLC, SDS-PAGE electrophoresis, and MALDI-TOF, respectively.
基金supported by the National 863 Program(No.2006AA02Z227)the Foundation of Key Subject Construct of Analytical Chemistry in Shaanxi Provincethe Foundation of Key Laboratory of Modem Separation Science in Shaanxi Province(No.05JS62).
文摘The refolding of the reduced/denatured insulin from bovine pancreas as the model protein was investigated with weak anion exchange chromatography (WAX) coupled with MALDI-TOF MS. The results indicated that the disulfide bonds almost cannot be formed correctly with the common mobile phase by WAX. However, with the urea gradient elution and in the presence of GSSG/ Cyst as the ratio 1:6 in the mobile phase employed, the disulfide exchange of reduced/denatured insulin can be accelerated resulting in forming the correct three disulfide bonds. The protein refolding efficiency of reduced/denatured insulin can be increased from 3 % to 34%. The effects of urea gradient and the oxidant and reductant groups, such as GSSG/GSH, Cyst, and GSSG/Cyst, on the forming the disulfide bonds of reduced/denatured insulin were investigated in detail. The results were further tested by the separation of the WAX fraction of reduced/denatured insulin with RPLC and MALDI-TOF MS.
文摘The thermal behaviors of five proteins in hydrophobic interaction chromatography (HIC) were investigated in the temperature range from 0 to 50℃. The thermodynamic parameters (△H°,△S°, △Cp°and △G°) of these proteins in the process of retention and unfolding were determined. The existence of enthalpy and entropy convergence with temperature was confirmed. The differences of the isoentropic and isoenthalpic temperatures for protein unfolding in HIC system from the traditional solution were elucidated.
基金the National Natural Science Foundation of China (Grant No. 20475042)
文摘A new method with only a single chromatographic column to accomplish the separation of intact protein molecules by the combination of ion exchange chromatography and hydrophobic interaction chromatography is firstly presented. By selecting twice a suitable stationary phase, mobile phase, buffer exchange condition, and sample injection, some proteins usually required to separate with two-dimensional chromatography can now be accomplished only using the single column in one hour. In addition, the separated proteins can maintain their original three-or four-dimensional molecular structure. It would be expected that this method can also provide a new thought for sample pre-frac-tionation in proteomic investigation, especially, for the limitation of sample source.
基金This work was supported by the National Natural Science Foundation of China (Grant Nos. 29675017 and 39180016).
文摘Substantial attention has been paid to the stoi-chiometric displacement models for solute retention (SDM-R) in liquid chromatography and adsorption (SDM-A) at surfaces since they were developed. The SDM has a strongly theoretical basis and has evolved to the point that it is widely applied in chemistry, biochemistry, molecular biology and biotechnology. This review introduces the history, recent developments, and new concepts relating to the SDM, including theory, mathematical expressions, and applications across a broad range of fields.
基金the National Natural Science Foundation in China(No.20705028)the Foundation of Key Laboratory of Modem Separation Science in Shaanxi Province(No.05JS61).
文摘Artificial molecular chaperone (AMC) and ion exchange chromatography (IEC) were integrated, thus a new refolding method, artificial molecular chaperone-ion exchange chromatography (AMC-IEC) was developed. Compared with AMC and IEC, the activity recovery of lysozyme obtained by AMC-IEC was much higher in the investigated range of initial protein concentrations, and the results show that AMC-IEC is very efficient for protein refolding at high concentrations. When the initial concentration of lysozyme is 180 mg/mL, its activity recovery obtained by AMC-IEC is still as high as 76.6%, while the activity recoveries obtained by AMC and IEC are 45.6% and 42.4%, respectively.
基金Project supported by the National Natural Science Foundation of China (Nos. 39880003 and 20175016 ).
文摘The monodisperse poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads with macroporous in the range of 8.0—12.0 m were prepared by a single-step swelling and polymerization method. The seed particles prepared by dispersion polymerization exhibited good absorption of the monomer phase. The pore size distribution of the beads was evaluated by gel permeation chromatography and mercury intrusion method. By using this media, a weak cation exchange (WCX) stationary phase for HPLC was synthesized by a new chemical modification method. The prepared resin has advantages of biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery and good resolution for proteins. The measured bioactivity recovery for lysozyme was (96±5)%. The dynamic protein loading capacity of the synthesized WCX packings was 21.3 mg/g. Five proteins were completely separated in 8.0 min using the synthesized WCX stationary phase. The experimental results show that the obtained WCX resin has very weak hydrophobicity. The WCX resin was also used for the rapid separation and purification of lysozyme from egg white in 8 min with only one step . The purity and specific bioactivity of the purified lysozyme was found more than 92.0% and 70184 U/mg, respectively.
基金supported by the National Natural Science Foundation(No.21006077)the foundation of science and technology in Shaanxi Province(No.2010K12-01-05)the foundation for construction of key disciplines of analytical chemistry in Shaanxi Province
文摘In this work, a kind of preparation method of zwitterionic ion chromatography (ZIC) stationary phase modified with phosphorylcholine (PC) was obtained by hydrolyzing after bonding phosphorylcholine dichloride to diol-silica to better explore the characteristics of the PC groups as ZIC stationary phase ligand in simultaneous separation of acidic proteins and basic proteins. The results showed that tv^o kinds of acidic proteins and three kinds of basic proteins can be separated completely, meanwhile, hen egg white was separated and purified and three kinds of egg white components ovalbumin, G2 ovoglobulin and ovotransfemin proteins were separated completely by one single step on PC-ZIC column, the purity of all proteins reached above 95%. PC-ZIC stationary phase was successfully improved with better separation capacity and selectivity than previously reported in this paper.
基金supported by the Modern Separation Science in Shaanxi Provincial Key Laboratory Foundation (09JS092)the Shaanxi Provin-cial Construction of Key Disciplines of Analytical Chemistry Foundation (05JS60)
文摘A new approach for separation of intact proteins with high resolution, high speed and high sample loading ("three highs") by non-porous packings in high performance liquid chromatography is presented.A chromatographic cake having larger diameter than its thickness is firstly employed to completely separate 1.0 and 40 μg of the mixture of 7 standard proteins within 1 min un-der conventional chromatographic conditions.Also, 0.5 mg of the mixture was almost completely separated within 1 min, accom-plishing the "three high" purpose.This research expands the application utilized for separation of intact proteins with non-porous packings.A smaller geometric size of chromatographic cake packed with much smaller particles of non-porous packings may improve results.
基金supported by the National 863 Program(No2006AA02Z227)the Natural Science Fondation of Shaanxi Province(No2011JZ002)the National Fund for Fostering Talents in Basic Science(NoJ0830417)
基金supported by the Foundation of Provincial Key Laboratory of Modern Separation Science (12JS091, 13JS117, 14JS096)
文摘Minor-adjustment of the retention of peptides, induced by varying the mobile phase flow-rate(MPF-R), is a new dynamic separation method for simultaneously and rapidly identifying and improving the selectivity of hidden and overlapping peptide peaks. It can also-stabilize the reverse elution order of some pair-peaks under gradient elution in reverse phase liquid chromatography. The retention characteristics of peptides under gradient elution in RPLC was firstly found to be dominated by two variables of the steady region(SR) and migration region(MR). The changes in peptide retention induced by varying the MPF-R can be attributed to changes in the rate of bond breaking of multiple molecular interactions of peptides from the SR and of the mass transfer of peptides from the stationary phase to the mobile phase in the MR. The two dynamic variables were also found to independently depend on the type of peptide. Desirable results were obtained using six standard oligopeptides and a real sample of trypsin-digested lysozyme.It is expected that the quality control of peptide drugs, high dispersion of peptide peaks in peptide mapping and "bottom-up MS"in proteomics will be improved by this method, even enabling peptide purification on a preparative scale in industry.
基金supported by the National Natural Science Foundation of China(No.39880003 and 20175016).
文摘The addition of packing material for high performance hydrophobic interaction chromatograghy (HPHIC) into the denaturant solution to prevent, or depress protein aggregation in the denatuaration process is presented. The renaturation of α-chymotrypsin (α-Chy) denatured with guanidine hydrochloride (GuHCl) solution indicated that renaturation efficiency can be enhanced from 36.1% to 59.0% by this new method. The structure of the ligand linking of HPHIC packings is also important for the protein renaturation.
基金Project supported by the National Natural Science Foundation of China (Nos. 39880003, 20175016).
文摘Based on the monodisperse poly(glycidyl methacrylate-co-ethylenedimethacrylate) beads (PGMA/EDMA) with macropore as a medium, a new hydrophilic medium cation exchange (MCX) stationary phase for HPLC was synthesized by a new chemically modified method. The stationary phase was evaluated with the property of ion exchange, separability, reproducibility, hydrophilicity, effect of salt concentration, salt types, column loading and pH on the separation and retention of proteins in detail. It was found that it follows ion exchange chromatographic (IEC) retention mechanism. The measured bioactivity recovery for lysozyme was (96 ± 5)%. The dynamic protein loading capacity of the synthesized MCX packings was 21.8 mg/g. Five proteins were almost completely separated within 6.0 min at a flow rate of 4 mL/min using the synthesized MCX resin. The MCX resin was also used for the rapid separation and purification of lysozyme from egg white with only one step. The purity and specific bioactivity of the purified lysozyme was found more than 95% and 70345 U/mg, respectively.
基金supported by the National 863 Program(No.2006AA02Z227)Natural Science Foundation of Shaanxi Province(No.2011JZ002)+1 种基金the Foundation of Key Laboratory in Shaanxi Province(Nos.2010JS103,11JS097,14JS098)Shaanxi Provincial Science and Technology Co-ordinating innovation projects(No.2013SZS18-K01)
文摘Ionic liquids(ILs) immobilized on silica as novel high performance liquid chromatography(HPLC)stationary phases have attracted considerable attention. However, it has not been applied to protein separation. In this paper, N-methylimidazolium IL-modified silica-based stationary phase(Silpr Mim)was prepared and investigated as a novel multi-interaction stationary phase charged positively for protein separation. The results indicate that all of the basic proteins tested cannot be absorbed on this novel stationary phase, whereas all of the acidic proteins tested can be retained, and the baseline separation of eight kinds of acidic protein standards can be achieved when performed in reversed phase/ion-exchange chromatography(RPLC/IEC) mode. Compared with commonly used commercial octadecylated silica(ODS) column, the novel stationary phase can show selectivity and good resolution to acidic proteins, which has a promising application in the separation and analyses of acidic proteins from the complex samples in proteomics. In addition, the chromatographic behavior of proteins, the effect of the ligand structure and the retention mechanism on this stationary phase were also investigated.
基金supported by the National Natu-ral Science Foundation of China(Grant Nos.39880003&20175016).
文摘The renaturation of the denatured α-chymotrypsin (α-Chy) with 1.7 mol · L-1 guanidine hydrochloride (GuHCI) by three kinds of stationary phase of high performance hydrophobic interaction chromatography (STHIC) with a comparable hydrophobicity but different ligand structures was investigated. The obtained result indicates that the ligand structures of the three STHIC contribute to the renaturation efficiency of α-Chy in the order of the end ligands PEG-600< phenyl group < tetrahydrofurfuryl alcohol (THFA).
基金ProjectsupportedbytheNationalNaturalScienceFoundationofChina (No .2 0 175 0 16)
文摘In previous paper a new adsorption isotherm which relates the amount of solute absorbed to the solvent concentration is proposed and simplified, and it only can be used at lower solute concentration. In this article the scope of the new adsorption isotherm is extended and the expressions with three and four parameters are obtained. The equations with multi parameters are valid when the adsorbed amounts are larger and show non linear logarithmic relationships. Tests with a homologue of aromatic alcohols by frontal analysis in reversed phase liquid chromatography demonstrate that the experimental results fit those equations well. In addition, the predicted values by the multi parameters were found to fit the experimental values well also. The parameters have physical meaning only for the two parameter equation for the aromatic alcohols.
基金supported by the National Natural Science Foundation of China(Grant Nos.29675017&39880003)the Foundation of the Young Teacher in the University of Ministry of Education,China(Grant No.DF00308)+1 种基金the Natural Science Foundation in Shaanxi Province(Grant No.2001H02)the Foundation of the Committee of Education in Shaanxi Province(Grant No.99JK101).
文摘The refolding of the reduced-denatured insulin from bovine pancreas was investigated with the size exclusion chromatography (SEC). It was shown that the reduced-denatured insulin originally denatured with 7.0 mol·L-1 guanidine hydrochloride (GuHCl) or 8.0 mol·L-1 urea could not be refolded with a non-oxidized mobile phase. Although the oxidized and reduced glutathione (GSSG and GSH) were employed in the oxidized mobile phase, the reduced-denatured insulin still could not be renatured. However, in the presence of 2.0 mol·L-1 urea in the oxidized mobile phase employed, the reduced-denatured insulin can be refolded with SEC, and the aggregation of denatured insulin can be diminished by urea. In addition, the disulfide exchange of reduced-denatured insulin also can be accelerated with GSSG/GSH in the oxidized mobile phase. The three disulfide bridges of insulin were formed correctly and the reduced-unfolded insulin can be renatured completely. The results were further tested with reversed-phase liquid chromatography (RPLC) and hydrophobic interaction chromatography (HIC).
文摘With frontal analysis (FA), the dependence of adsorption isotherms of insulin on the composition of mobile phase in reversed phase liquid chromatography (RPLC) has been investigated. This is also a good example to employ the stoichiometric displacement theory (SDT) for investigating solute adsorption in physical chemistry. Six kinds of mobile phase in RPLC were employed to study the effects on the elution curves and adsorption isotherms of insulin. The key points of this paper are: (1) The stability of insulin due to delay time after preparing, the organic solvent concentration, the kind and the concentration of ion pairing agent in mobile phase were found to affect both elution curve and adsorption isotherm very seriously. (2) To obtain a valid and comparable result, the composition of the mobile phase employed in FA must be as same as possible to that in usual RPLC of either analytical scale or preparative purpose. (3) Langmuir Equation and the SDT were employed to imitate these obtained adsorption isotherms. The expression for solute adsorption from solution of the SDT was found to have a better elucidation to the insulin adsorption from mobile phase in RPLC.