The contributions of tetrahydrofurfuryl alcohol (THFA) and polyethylene glycol (PEG) to the renatured efficiency of a-chymotrypsin were investigated and compared with each other. The maximum increments of bioactivity...The contributions of tetrahydrofurfuryl alcohol (THFA) and polyethylene glycol (PEG) to the renatured efficiency of a-chymotrypsin were investigated and compared with each other. The maximum increments of bioactivity recovery of a-Chy were found to be 25.1% for THFA, 10.4% for PEG, respectively. The experimental results indicated that the denaturant solution containing THFA contributed more to the renaturation of a-Chy in high performance hydrophobic interaction chromatography (HPHIC) than that containing PEG, when the concentration of THFA was 3.2%, the bioactivity recovery of a-Chy is the highest.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
In this work,a new type of hydrophobic stationary phase that provide electrostatic interactions with analytes was developed by bondingβ-phenylethylamine as a functional ligand to silica.This stationary phase can sepa...In this work,a new type of hydrophobic stationary phase that provide electrostatic interactions with analytes was developed by bondingβ-phenylethylamine as a functional ligand to silica.This stationary phase can separate proteins with similar hydrophobicity that traditional hydrophobic resins cannot.Hen egg white was separated to examine the selectivity.The results show that the introduced electrostatic interactions are an important factor for the resolution enhancement and the new resin could have important applications in separation and purification of biological macromolecules.展开更多
A unified retention equation of proteins was proved to be valid for a mixed-mode interaction mechanism in ion exchange chromatography (IEC) and hydrophobia interaction chro-matography (HIC). The reason to form a '...A unified retention equation of proteins was proved to be valid for a mixed-mode interaction mechanism in ion exchange chromatography (IEC) and hydrophobia interaction chro-matography (HIC). The reason to form a 'U' shape retention curve of proteins hi both HIC and IEC was explained and the concentration range of the strongest elution ability for the mobile phase was determined with this equation. The parameters in this equation could be used to characterize the difference for either HIC or IEC adsorbents and the changes in the molecular conformation of proteins. With the parameters in this equation, the contributions of salt and water in the mobile phase to the protein retention in HIC and IEC were discussed, respectively. In addition, the comparison between the unified equation and Melander' s three-parameter equation for mixed-mode interaction chromatography was also investigated and better results were obtained in former equation.展开更多
Hydrophobic interaction chromatography （HIC） is a rapid growing bioseparation technique, which separates biomolecules, such as therapeutic proteins and antibodys, based on the reversible hydrophobic interaction betw...Hydrophobic interaction chromatography （HIC） is a rapid growing bioseparation technique, which separates biomolecules, such as therapeutic proteins and antibodys, based on the reversible hydrophobic interaction between immobilized hydrophobic ligands on chromatographic resin spheres and non-polar regions of solute molecule. In this review, the fundamental concepts of HIC and the factors that may affect purification efficiency of HIC is summarized, followed by the comparison of HIC with affinity chromatography and ion-exchange chromatography. Hydrophobic interaction membrane chromatography （HIMC） combines the advantages of HIC and membrane process and has showed great potential in bioseparation. For better understanding of HIMC, this review presents an overview of two main concerns about HIMC, i.e. membrane materials and hydrophobic ligands. Specifically, cellulose fiber-based membrane substrate and environment-responsive ligands are emphasized.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 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
Hydrophobic interaction chromatography(HIC)as an indispensable method for protein purification has attracted considerable attentions of researchers as well as biopharmaceutical industries.However,the low binding capac...Hydrophobic interaction chromatography(HIC)as an indispensable method for protein purification has attracted considerable attentions of researchers as well as biopharmaceutical industries.However,the low binding capacity and slow adsorption rate of the currently available HIC media lead to a little supply and high price of the highly purified proteins.Herein,nanofibrous membranes with hydrophobic binding sites were developed for HIC by directly coupling phenyl glycidyl ether on the hydrolyzed cellulose acetate nanofiber membrane(cellulose-phenyl NFM).Scanning electron microscope(SEM),water contact angle(WCA),Fourier transform infrared(FTIR),thermogravimetric analysis(TGA),Brunauer-Emmett-Teller(BET)surface area analysis and capillary flow porometer(CFP)were applied to evaluate the physically and chemically structural transformation.The obtained cellulose-phenyl NFMs showed a proper hydrophilcity(WCA=37°),a relatively high BET surface area(3.6 times the surface area of commercial fibrous membranes),and tortuous-channel structure with through-hole size in the range of 0.25-1.2μm,which led to a little non-specificity adsorption,high bovine serum albumin adsorption capacity of 118 mg g^(−1),fast adsorption process within 12 h,good long-term stability and reusability.Moreover,compared with traditional modification methods which always include activation and graft two steps,direct coupling method is more efficient for HIC media fabrication.Therefore,cellulose-phenyl NFMs with outstanding protein adsorption performance could be a kind of promising candidate for HIC.展开更多
Protein expression in E coil often results in the formation of a kind of protein aggregate called inclusion body Conversion of the inactive protein aggregate into biologically active protein is a key step in productio...Protein expression in E coil often results in the formation of a kind of protein aggregate called inclusion body Conversion of the inactive protein aggregate into biologically active protein is a key step in production of recombinant products Convenlional dilution refolding technique suffers from disadvantages of low recovery and low concentration Various chromatographic refolding techniques have been developed over the last few years These include size-exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography and different affinity chromatography. A successful strategy is the use of gradient elution in column control which provides a gentle and gradual change of the solution environment for the macromolecule to rsfold at nano-scale, The gradient refolding at column scale could minimize misfolding and aggregation which are induced by sudden change of the solution in conventional refolding operation.展开更多
基金These projects No. 39880003 and 20175016 were supported by the National Natural Science Foundation of China.
文摘The contributions of tetrahydrofurfuryl alcohol (THFA) and polyethylene glycol (PEG) to the renatured efficiency of a-chymotrypsin were investigated and compared with each other. The maximum increments of bioactivity recovery of a-Chy were found to be 25.1% for THFA, 10.4% for PEG, respectively. The experimental results indicated that the denaturant solution containing THFA contributed more to the renaturation of a-Chy in high performance hydrophobic interaction chromatography (HPHIC) than that containing PEG, when the concentration of THFA was 3.2%, the bioactivity recovery of a-Chy is the highest.
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supported by the National Natural Science Foundation in China(No.21006077)the Foundation of Northwest University(No.09NW14)
文摘In this work,a new type of hydrophobic stationary phase that provide electrostatic interactions with analytes was developed by bondingβ-phenylethylamine as a functional ligand to silica.This stationary phase can separate proteins with similar hydrophobicity that traditional hydrophobic resins cannot.Hen egg white was separated to examine the selectivity.The results show that the introduced electrostatic interactions are an important factor for the resolution enhancement and the new resin could have important applications in separation and purification of biological macromolecules.
基金Project (Nos. 296750l7 and 39880003) supported by the National Natural Science Foundation of China.
文摘A unified retention equation of proteins was proved to be valid for a mixed-mode interaction mechanism in ion exchange chromatography (IEC) and hydrophobia interaction chro-matography (HIC). The reason to form a 'U' shape retention curve of proteins hi both HIC and IEC was explained and the concentration range of the strongest elution ability for the mobile phase was determined with this equation. The parameters in this equation could be used to characterize the difference for either HIC or IEC adsorbents and the changes in the molecular conformation of proteins. With the parameters in this equation, the contributions of salt and water in the mobile phase to the protein retention in HIC and IEC were discussed, respectively. In addition, the comparison between the unified equation and Melander' s three-parameter equation for mixed-mode interaction chromatography was also investigated and better results were obtained in former equation.
基金Acknowledgement The authors would like to thank the National Natural Science Foundation of China （Grant No. 20874004） for financial support.
文摘Hydrophobic interaction chromatography （HIC） is a rapid growing bioseparation technique, which separates biomolecules, such as therapeutic proteins and antibodys, based on the reversible hydrophobic interaction between immobilized hydrophobic ligands on chromatographic resin spheres and non-polar regions of solute molecule. In this review, the fundamental concepts of HIC and the factors that may affect purification efficiency of HIC is summarized, followed by the comparison of HIC with affinity chromatography and ion-exchange chromatography. Hydrophobic interaction membrane chromatography （HIMC） combines the advantages of HIC and membrane process and has showed great potential in bioseparation. For better understanding of HIMC, this review presents an overview of two main concerns about HIMC, i.e. membrane materials and hydrophobic ligands. Specifically, cellulose fiber-based membrane substrate and environment-responsive ligands are emphasized.
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 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This work was supported by the National Natural Science Foundation of China(Grant Nos.51673037,51873029,and 81771338)the Science and Technology Commission of Shanghai Municipality(Grant No.18511109500).
文摘Hydrophobic interaction chromatography(HIC)as an indispensable method for protein purification has attracted considerable attentions of researchers as well as biopharmaceutical industries.However,the low binding capacity and slow adsorption rate of the currently available HIC media lead to a little supply and high price of the highly purified proteins.Herein,nanofibrous membranes with hydrophobic binding sites were developed for HIC by directly coupling phenyl glycidyl ether on the hydrolyzed cellulose acetate nanofiber membrane(cellulose-phenyl NFM).Scanning electron microscope(SEM),water contact angle(WCA),Fourier transform infrared(FTIR),thermogravimetric analysis(TGA),Brunauer-Emmett-Teller(BET)surface area analysis and capillary flow porometer(CFP)were applied to evaluate the physically and chemically structural transformation.The obtained cellulose-phenyl NFMs showed a proper hydrophilcity(WCA=37°),a relatively high BET surface area(3.6 times the surface area of commercial fibrous membranes),and tortuous-channel structure with through-hole size in the range of 0.25-1.2μm,which led to a little non-specificity adsorption,high bovine serum albumin adsorption capacity of 118 mg g^(−1),fast adsorption process within 12 h,good long-term stability and reusability.Moreover,compared with traditional modification methods which always include activation and graft two steps,direct coupling method is more efficient for HIC media fabrication.Therefore,cellulose-phenyl NFMs with outstanding protein adsorption performance could be a kind of promising candidate for HIC.
基金The Natural Science Foundation of China(NSFC No.20136020,20125616)Chinese Academy of Sciences are gratefully acknowledged for financial supports to this research.
文摘Protein expression in E coil often results in the formation of a kind of protein aggregate called inclusion body Conversion of the inactive protein aggregate into biologically active protein is a key step in production of recombinant products Convenlional dilution refolding technique suffers from disadvantages of low recovery and low concentration Various chromatographic refolding techniques have been developed over the last few years These include size-exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography and different affinity chromatography. A successful strategy is the use of gradient elution in column control which provides a gentle and gradual change of the solution environment for the macromolecule to rsfold at nano-scale, The gradient refolding at column scale could minimize misfolding and aggregation which are induced by sudden change of the solution in conventional refolding operation.
