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
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 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
Recombinant human granulocyte colony-stimulating factor (rhG-CSF) in inclusion bodies was solubilized by 8 mol/L urea solution and subsequently precipitated by acetone to improve its purity. After that, the precipit...Recombinant human granulocyte colony-stimulating factor (rhG-CSF) in inclusion bodies was solubilized by 8 mol/L urea solution and subsequently precipitated by acetone to improve its purity. After that, the precipitates were solubilized by sodium hydroxide solution containing 2 mol/L urea. Then the solubilized rhG-CSF was passed through a size exclusion chromatography for refolding and extensive purification, and further purified by a weak anion exchange chromatography. The purity and mass recovery of refolded rhG-CSF were 96.5% and 75.6%, respectively. The bioactivity was 8.4x10^7 IU/mg.展开更多
Trehalose,a unique nonreducing crystalline disaccharide,is a potential disease-modifying treatment for neurodegenerative diseases associated with protein misfolding and aggregation due to aging,intrinsic mutations,or ...Trehalose,a unique nonreducing crystalline disaccharide,is a potential disease-modifying treatment for neurodegenerative diseases associated with protein misfolding and aggregation due to aging,intrinsic mutations,or autophagy dysregulation.This systematic review summarizes the effects of trehalose on its underlying mechanisms in animal models of selected neurodegenerative disorders(tau pathology,synucleinopathy,polyglutamine tract,and motor neuron diseases).All animal studies on neurodegenerative diseases treated with trehalose published in Medline(accessed via EBSCOhost)and Scopus were considered.Of the 2259 studies screened,29 met the eligibility criteria.According to the SYstematic Review Center for Laboratory Animal Experiment(SYRCLE)risk of bias tool,we reported 22 out of 29 studies with a high risk of bias.The present findings support the purported role of trehalose in autophagic flux and protein refolding.This review identified several other lesser-known pathways,including modifying amyloid precursor protein processing,inhibition of reactive gliosis,the integrity of the blood-brain barrier,activation of growth factors,upregulation of the downstream antioxidant signaling pathway,and protection against mitochondrial defects.The absence of adverse events and improvements in the outcome parameters were observed in some studies,which supports the transition to human clinical trials.It is possible to conclude that trehalose exerts its neuroprotective effects through both direct and indirect pathways.However,heterogeneous methodologies and outcome measures across the studies rendered it impossible to derive a definitive conclusion.Translational studies on trehalose would need to clarify three important questions:1)bioavailability with oral administration,2)optimal time window to confer neuroprotective benefits,and 3)optimal dosage to confer neuroprotection.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.
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.
基金This work is supported by the National Natural Science Foundation of china (No. 20175016 and No. 20475042) the Foundation of Key Laboratory of Modem Separation Science in Shaanxi Province (No. 05JS61).
文摘Recombinant human granulocyte colony-stimulating factor (rhG-CSF) in inclusion bodies was solubilized by 8 mol/L urea solution and subsequently precipitated by acetone to improve its purity. After that, the precipitates were solubilized by sodium hydroxide solution containing 2 mol/L urea. Then the solubilized rhG-CSF was passed through a size exclusion chromatography for refolding and extensive purification, and further purified by a weak anion exchange chromatography. The purity and mass recovery of refolded rhG-CSF were 96.5% and 75.6%, respectively. The bioactivity was 8.4x10^7 IU/mg.
基金supported by Dana Impak Perdana Grant(DIP-2019-007)received by NMI from Universiti Kebangsaan Malaysia.
文摘Trehalose,a unique nonreducing crystalline disaccharide,is a potential disease-modifying treatment for neurodegenerative diseases associated with protein misfolding and aggregation due to aging,intrinsic mutations,or autophagy dysregulation.This systematic review summarizes the effects of trehalose on its underlying mechanisms in animal models of selected neurodegenerative disorders(tau pathology,synucleinopathy,polyglutamine tract,and motor neuron diseases).All animal studies on neurodegenerative diseases treated with trehalose published in Medline(accessed via EBSCOhost)and Scopus were considered.Of the 2259 studies screened,29 met the eligibility criteria.According to the SYstematic Review Center for Laboratory Animal Experiment(SYRCLE)risk of bias tool,we reported 22 out of 29 studies with a high risk of bias.The present findings support the purported role of trehalose in autophagic flux and protein refolding.This review identified several other lesser-known pathways,including modifying amyloid precursor protein processing,inhibition of reactive gliosis,the integrity of the blood-brain barrier,activation of growth factors,upregulation of the downstream antioxidant signaling pathway,and protection against mitochondrial defects.The absence of adverse events and improvements in the outcome parameters were observed in some studies,which supports the transition to human clinical trials.It is possible to conclude that trehalose exerts its neuroprotective effects through both direct and indirect pathways.However,heterogeneous methodologies and outcome measures across the studies rendered it impossible to derive a definitive conclusion.Translational studies on trehalose would need to clarify three important questions:1)bioavailability with oral administration,2)optimal time window to confer neuroprotective benefits,and 3)optimal dosage to confer neuroprotection.
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.