The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Yan Sun - One of the best experts on this subject based on the ideXlab platform.
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Protein behavior at surfaces orientation conformational transitions and transport
Journal of Chromatography A, 2015Co-Authors: Lin Zhang, Yan SunAbstract:Chromatography is the key technology in Protein purification as well as in Protein Refolding. Taking the scientific development and technological innovation of Protein chromatography as the objective, this article is devoted to an overview of Protein behavior at chromatographic surfaces, including Protein orientation, conformational transitions (unfolding and Refolding), and Protein transport. Recent advances achieved by using molecular simulations as well as theoretical and experimental investigations are elaborated and discussed with emphasis on their implications to the rational design of novel chromatographic surfaces or materials and mobile phase conditions for the development of high-performance Protein chromatography.
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a double modification strategy for enhancing charge density of mono sized beads for facilitated Refolding of like charged Protein
Journal of Chromatography A, 2013Co-Authors: Chunyan Yang, Xiaoyan Dong, Yan SunAbstract:We have previously found that addition of like-charge media in a Refolding system can greatly enhance Protein Refolding yield, and the media of higher charge density (q) are favorable for facilitating Protein Refolding. Herein, a double-modification strategy was developed to increase the charge density of mono-sized poly(glycidyl methacrylate) (PGMA) microspheres (0.8μm). The PGMA beads were firstly modified with poly(ethylenimine) of molecular weight 60,000 (PEIL) or 1200 (PEIS) and then further modified with 2-diethylaminoethylchloride (DEAE) or PEIS. The charge density and bovine serum albumin (BSA) adsorption density (QBSA) of the beads were significantly increased by the double modification. The QBSA of the double-modified beads with the highest q (824μmol/g) reached 36.3mg/g, over 35% higher than that of the single-modified beads (26.7mg/g) with the highest q (300μmol/g) that a single modification could achieve. The double-modified beads with different ligand structures and charge densities were used for facilitating the Refolding of like-charged lysozyme. Lysozyme Refolding yield with the single-modified beads of the highest q (300μmol/g) was 60% at a critical bead concentration (cc) of 100mg/mL. By contrast, the Refolding yields with the double-modified beads of q>500μmol/g were 70%, and the Refolding yields with the double-modified beads of q≥650μmol/g could even reach 70% at a cc of 40mg/mL. This indicates that the double-modified beads of high q values could enhance like-charged Protein Refolding more significantly than the single-modified beads at a low bead utilization. The facilitating effect of the like-charged beads was independent of the ligand structure. The beads of higher q values showed lower salt sensitivity in Protein Refolding, and were expected beneficial for use in Refolding buffers of higher ionic strengths. The sequential modification strategy for enhancing charge density would help develop more efficient media for Protein adsorption and Protein Refolding applications.
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ion exchange resins facilitate like charged Protein Refolding effects of porous solid phase properties
Journal of Chromatography A, 2012Co-Authors: Xiaoyan Dong, Yan SunAbstract:We have previously found that addition of charged particles in a Refolding solution can greatly increase the Refolding yield of like-charged Proteins. Herein, porous anion exchangers of different charged group densities, ligand chemistries, pore sizes and particle sizes were prepared with Sepharose FF gel for studying their effects on the oxidative Refolding of like-charged lysozyme. We found that charge density had significant contribution to the enhancing effects on lysozyme Refolding. At low resin concentration range (<0.04-0.1g/mL), the Refolding yield increased with increasing charged group density and resin concentration. The yield then reached a plateau at a critical resin concentration; the higher the charge density, the lower the critical resin concentration. This implies that gel particles of higher charge density were favorable to offer higher Refolding yield at lower added concentrations. In the gel concentration range in which Refolding yield has reached plateau, there existed an optimum charge density that gave the highest Refolding yield. It was attributed to the electrostatic repulsion effect of the charged groups on the like-charged Protein, which reduced the accessible pore volume for the Protein. At the same charge density, the Refolding yield was independent of ligand chemistry, but a polyelectrolyte group of higher molecular weight was more suitable for grafting the gel to prepare matrices of high charge density. The resins of smaller size exhibited better facilitating effect, and the microporous resin was better than that with superpores. The research is expected to help design more effective charged materials for facilitating Protein Refolding.
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ion exchange resins facilitate like charged Protein Refolding effects of porous solid phase properties
Journal of Chromatography A, 2012Co-Authors: Xiaoyan Dong, Yan SunAbstract:Abstract We have previously found that addition of charged particles in a Refolding solution can greatly increase the Refolding yield of like-charged Proteins. Herein, porous anion exchangers of different charged group densities, ligand chemistries, pore sizes and particle sizes were prepared with Sepharose FF gel for studying their effects on the oxidative Refolding of like-charged lysozyme. We found that charge density had significant contribution to the enhancing effects on lysozyme Refolding. At low resin concentration range (
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ion exchange resins greatly facilitate Refolding of like charged Proteins at high concentrations
Biotechnology and Bioengineering, 2011Co-Authors: Guozhen Wang, Xiaoyan Dong, Yan SunAbstract:Protein Refolding is a crucial step for the production of therapeutic Proteins expressed in bacteria as inclusion bodies. In vitro Protein Refolding is severely impeded by the aggregation of folding intermediates during the folding process, so inhibition of the aggregation is the most effective approach to high-efficiency Protein Refolding. We have herein found that electrostatic repulsion between like-charged Protein and ion exchange gel beads can greatly suppress the aggregation of folding intermediates, leading to the significant increase of native Protein recovery. This finding is extensively demonstrated with three different Proteins and four kinds of ion-exchange resins when the Protein and ion-exchange gel are either positively or negatively charged at the Refolding conditions. It is remarkable that the enhancing effect is significant at very high Protein concentrations, such as 4 mg/mL lysozyme (positively charged) and 2 mg/mL bovine serum albumin (negatively charged). Moreover, the folding kinetics is not compromised by the presence of the resins, so fast Protein Refolding is realized at high Protein concentrations. It was not realistic by any other approaches. The working mechanism of the like-charged resin is considered due to the charge repulsion that could induce oriented alignment of Protein molecules near the charged surface, leading to the inhibition of Protein aggregation. The molecular crowding effect induced by the charge repulsion may also contribute to accelerating Protein folding. The Refolding method with like-charged ion exchangers is simple to perform, and the key material is easy to separate for recycling. Moreover, because ion exchangers can work as adsorbents of oppositely charged impurities, an operation of simultaneous Protein Refolding and purification is possible. All the characters are desirable for preparative Refolding of therapeutic Proteins expressed in bacteria as inclusion bodies. Bioeng. 2011; 108:1068–1077. © 2010 Wiley Periodicals, Inc.
Ary A Hoffmann - One of the best experts on this subject based on the ideXlab platform.
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stress responses of small heat shock Protein genes in lepidoptera point to limited conservation of function across phylogeny
PLOS ONE, 2015Co-Authors: Bo Zhang, Jincheng Zheng, Yu Peng, Xiaoxia Liu, Ary A HoffmannAbstract:The small heat shock Protein (sHsp) family is thought to play an important role in Protein Refolding and signal transduction, and thereby protect organisms from stress. However little is known about sHsp function and conservation across phylogenies. In the current study, we provide a comprehensive assessment of small Hsp genes and their stress responses in the oriental fruit moth (OFM), Grapholita molesta. Fourteen small heat shock Proteins of OFM clustered with related Hsps in other Lepidoptera despite a high level of variability among them, and in contrast to the highly conserved Hsp11.1. The only known lepidopteran sHsp ortholog (Hsp21.3) was consistently unaffected under thermal stress in Lepidoptera where it has been characterized. However the phylogenetic position of the sHsps within the Lepidoptera was not associated with conservation of induction patterns under thermal extremes or diapause. These findings suggest that the sHsps have evolved rapidly to develop new functions within the Lepidoptera.
Wangxia Wang - One of the best experts on this subject based on the ideXlab platform.
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role of plant heat shock Proteins and molecular chaperones in the abiotic stress response
Trends in Plant Science, 2004Co-Authors: Wangxia Wang, Basia Vinocur, Oded Shoseyov, Arie AltmanAbstract:Abstract Abiotic stresses usually cause Protein dysfunction. Maintaining Proteins in their functional conformations and preventing the aggregation of non-native Proteins are particularly important for cell survival under stress. Heat-shock Proteins (Hsps)/chaperones are responsible for Protein folding, assembly, translocation and degradation in many normal cellular processes, stabilize Proteins and membranes, and can assist in Protein Refolding under stress conditions. They can play a crucial role in protecting plants against stress by re-establishing normal Protein conformation and thus cellular homeostasis. Here, we summarize the significance of Hsps and chaperones in abiotic stress responses in plants, and discuss the co-operation among their different classes and their interactions with other stress-induced components.
Xiaoyan Dong - One of the best experts on this subject based on the ideXlab platform.
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a double modification strategy for enhancing charge density of mono sized beads for facilitated Refolding of like charged Protein
Journal of Chromatography A, 2013Co-Authors: Chunyan Yang, Xiaoyan Dong, Yan SunAbstract:We have previously found that addition of like-charge media in a Refolding system can greatly enhance Protein Refolding yield, and the media of higher charge density (q) are favorable for facilitating Protein Refolding. Herein, a double-modification strategy was developed to increase the charge density of mono-sized poly(glycidyl methacrylate) (PGMA) microspheres (0.8μm). The PGMA beads were firstly modified with poly(ethylenimine) of molecular weight 60,000 (PEIL) or 1200 (PEIS) and then further modified with 2-diethylaminoethylchloride (DEAE) or PEIS. The charge density and bovine serum albumin (BSA) adsorption density (QBSA) of the beads were significantly increased by the double modification. The QBSA of the double-modified beads with the highest q (824μmol/g) reached 36.3mg/g, over 35% higher than that of the single-modified beads (26.7mg/g) with the highest q (300μmol/g) that a single modification could achieve. The double-modified beads with different ligand structures and charge densities were used for facilitating the Refolding of like-charged lysozyme. Lysozyme Refolding yield with the single-modified beads of the highest q (300μmol/g) was 60% at a critical bead concentration (cc) of 100mg/mL. By contrast, the Refolding yields with the double-modified beads of q>500μmol/g were 70%, and the Refolding yields with the double-modified beads of q≥650μmol/g could even reach 70% at a cc of 40mg/mL. This indicates that the double-modified beads of high q values could enhance like-charged Protein Refolding more significantly than the single-modified beads at a low bead utilization. The facilitating effect of the like-charged beads was independent of the ligand structure. The beads of higher q values showed lower salt sensitivity in Protein Refolding, and were expected beneficial for use in Refolding buffers of higher ionic strengths. The sequential modification strategy for enhancing charge density would help develop more efficient media for Protein adsorption and Protein Refolding applications.
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ion exchange resins facilitate like charged Protein Refolding effects of porous solid phase properties
Journal of Chromatography A, 2012Co-Authors: Xiaoyan Dong, Yan SunAbstract:We have previously found that addition of charged particles in a Refolding solution can greatly increase the Refolding yield of like-charged Proteins. Herein, porous anion exchangers of different charged group densities, ligand chemistries, pore sizes and particle sizes were prepared with Sepharose FF gel for studying their effects on the oxidative Refolding of like-charged lysozyme. We found that charge density had significant contribution to the enhancing effects on lysozyme Refolding. At low resin concentration range (<0.04-0.1g/mL), the Refolding yield increased with increasing charged group density and resin concentration. The yield then reached a plateau at a critical resin concentration; the higher the charge density, the lower the critical resin concentration. This implies that gel particles of higher charge density were favorable to offer higher Refolding yield at lower added concentrations. In the gel concentration range in which Refolding yield has reached plateau, there existed an optimum charge density that gave the highest Refolding yield. It was attributed to the electrostatic repulsion effect of the charged groups on the like-charged Protein, which reduced the accessible pore volume for the Protein. At the same charge density, the Refolding yield was independent of ligand chemistry, but a polyelectrolyte group of higher molecular weight was more suitable for grafting the gel to prepare matrices of high charge density. The resins of smaller size exhibited better facilitating effect, and the microporous resin was better than that with superpores. The research is expected to help design more effective charged materials for facilitating Protein Refolding.
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ion exchange resins facilitate like charged Protein Refolding effects of porous solid phase properties
Journal of Chromatography A, 2012Co-Authors: Xiaoyan Dong, Yan SunAbstract:Abstract We have previously found that addition of charged particles in a Refolding solution can greatly increase the Refolding yield of like-charged Proteins. Herein, porous anion exchangers of different charged group densities, ligand chemistries, pore sizes and particle sizes were prepared with Sepharose FF gel for studying their effects on the oxidative Refolding of like-charged lysozyme. We found that charge density had significant contribution to the enhancing effects on lysozyme Refolding. At low resin concentration range (
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ion exchange resins greatly facilitate Refolding of like charged Proteins at high concentrations
Biotechnology and Bioengineering, 2011Co-Authors: Guozhen Wang, Xiaoyan Dong, Yan SunAbstract:Protein Refolding is a crucial step for the production of therapeutic Proteins expressed in bacteria as inclusion bodies. In vitro Protein Refolding is severely impeded by the aggregation of folding intermediates during the folding process, so inhibition of the aggregation is the most effective approach to high-efficiency Protein Refolding. We have herein found that electrostatic repulsion between like-charged Protein and ion exchange gel beads can greatly suppress the aggregation of folding intermediates, leading to the significant increase of native Protein recovery. This finding is extensively demonstrated with three different Proteins and four kinds of ion-exchange resins when the Protein and ion-exchange gel are either positively or negatively charged at the Refolding conditions. It is remarkable that the enhancing effect is significant at very high Protein concentrations, such as 4 mg/mL lysozyme (positively charged) and 2 mg/mL bovine serum albumin (negatively charged). Moreover, the folding kinetics is not compromised by the presence of the resins, so fast Protein Refolding is realized at high Protein concentrations. It was not realistic by any other approaches. The working mechanism of the like-charged resin is considered due to the charge repulsion that could induce oriented alignment of Protein molecules near the charged surface, leading to the inhibition of Protein aggregation. The molecular crowding effect induced by the charge repulsion may also contribute to accelerating Protein folding. The Refolding method with like-charged ion exchangers is simple to perform, and the key material is easy to separate for recycling. Moreover, because ion exchangers can work as adsorbents of oppositely charged impurities, an operation of simultaneous Protein Refolding and purification is possible. All the characters are desirable for preparative Refolding of therapeutic Proteins expressed in bacteria as inclusion bodies. Bioeng. 2011; 108:1068–1077. © 2010 Wiley Periodicals, Inc.
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new development of reverse micelles and applications in Protein separation and Refolding
Chinese Journal of Chemical Engineering, 2008Co-Authors: Yang Liu, Xiaoyan Dong, Yan SunAbstract:Abstract Reverse micelles bring mild and effective microenvironments in organic solvent that contain bio-molecules, which have attracted immense attention for application in the isolation of Proteins, Protein Refolding, and enzymatic reaction. In this review, the application of reverse micelles for Protein separation and Refolding has been briefly summarized and various reverse micellar systems composed of different surfactants, including ionic, nonionic, mixed, and affinity-based reverse micelles, have been highlighted. It illustrates especially the potential application of the novel affinity-based reverse micelles consisting of biocompatible surfactant coupled with affinity ligands. Moreover, the importance to develop universal affinity-based reverse micelles for Protein separation and Refolding in the downstream processing of biotechnology has been pointed out.
Krzysztof Liberek - One of the best experts on this subject based on the ideXlab platform.
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hsp70 displaces small heat shock Proteins from aggregates to initiate Protein Refolding
The EMBO Journal, 2017Co-Authors: Szymon żwirowski, Agnieszka Klosowska, Igor Obuchowski, Nadinath B Nillegoda, Artur Pirog, Szymon Zietkiewicz, Bernd Bukau, Axel Mogk, Krzysztof LiberekAbstract:Small heat shock Proteins (sHsps) are an evolutionary conserved class of ATP-independent chaperones that protect cells against proteotoxic stress. sHsps form assemblies with aggregation-prone misfolded Proteins, which facilitates subsequent substrate solubilization and Refolding by ATP-dependent Hsp70 and Hsp100 chaperones. Substrate solubilization requires disruption of sHsp association with trapped misfolded Proteins. Here, we unravel a specific interplay between Hsp70 and sHsps at the initial step of the solubilization process. We show that Hsp70 displaces surface-bound sHsps from sHsp-substrate assemblies. This Hsp70 activity is unique among chaperones and highly sensitive to alterations in Hsp70 concentrations. The Hsp70 activity is reflected in the organization of sHsp-substrate assemblies, including an outer dynamic sHsp shell that is removed by Hsp70 and a stable core comprised mainly of aggregated substrates. Binding of Hsp70 to the sHsp/substrate core protects the core from aggregation and directs sequestered substrates towards Refolding pathway. The sHsp/Hsp70 interplay has major impact on Protein homeostasis as it sensitizes substrate release towards cellular Hsp70 availability ensuring efficient Refolding of damaged Proteins under favourable folding conditions.
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mitochondrial hsp78 a member of the clp hsp100 family in saccharomyces cerevisiae cooperates with hsp70 in Protein Refolding
FEBS Letters, 2001Co-Authors: Joanna Krzewska, Thomas Langer, Krzysztof LiberekAbstract:The molecular chaperone Protein Hsp78, a member of the Clp/Hsp100 family localized in the mitochondria of Saccharomyces cerevisiae, is required for maintenance of mitochondrial functions under heat stress. To characterize the biochemical mechanisms of Hsp78 function, Hsp78 was purified to homogeneity and its role in the reactivation of chemically and heat-denatured substrate Protein was analyzed in vitro. Hsp78 alone was not able to mediate reactivation of firefly luciferase. Rather, efficient Refolding was dependent on the simultaneous presence of Hsp78 and the mitochondrial Hsp70 machinery, composed of Ssc1p/Mdj1p/Mge1p. Bacterial DnaK/DnaJ/GrpE, which cooperates with the Hsp78 homolog, ClpB in Escherichia coli, could not substitute for the mitochondrial Hsp70 system. However, efficient Hsp78-dependent Refolding of luciferase was observed if DnaK was replaced by Ssc1p in these experiments, suggesting a specific functional interaction of both chaperone Proteins. These findings establish the cooperation of Hsp78 with the Hsp70 machinery in the Refolding of heat-inactivated Proteins and demonstrate a conserved mode of action of ClpB homologs.
