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dc.contributor.authorGu, Zhenyu
dc.contributor.authorZhu, Xiaonan
dc.contributor.authorNi, Shaowei
dc.contributor.authorSu, Zhiguo
dc.contributor.authorZhou, Hai-Meng
dc.date2022-08-11T08:08:00.000
dc.date.accessioned2022-08-23T15:38:41Z
dc.date.available2022-08-23T15:38:41Z
dc.date.issued2004-03-10
dc.date.submitted2010-03-17
dc.identifier.citationInt J Biochem Cell Biol. 2004 May;36(5):795-805. <a href="http://dx.doi.org/10.1016/j.biocel.2003.08.015">Link to article on publisher's site</a>
dc.identifier.issn1357-2725 (Linking)
dc.identifier.doi10.1016/j.biocel.2003.08.015
dc.identifier.pmid15006632
dc.identifier.urihttp://hdl.handle.net/20.500.14038/25986
dc.description.abstractIt is believed that denatured-reduced lysozyme rapidly forms aggregates during refolding process, which is often worked around by operating at low protein concentrations or in the presence of aggregation inhibitors. However, we found that low concentration buffer alone could efficiently suppress aggregation. Based on this finding, stable equilibrium intermediate states of denatured-reduced lysozyme containing eight free SH groups were obtained in the absence of redox reagents in buffer of low concentrations alone at neutral or mildly alkaline pH. Transition in the secondary structure of the intermediate from native-like to beta-sheet was observed by circular dichroism (CD) as conditions were varied. Dynamic light scattering and ANS-binding studies showed that the self-association accompanied the conformational change and the structure rich in beta-sheet was the intermediate state for aggregation, which could form either amyloid protofibril or amorphous aggregates under different conditions as detected by Electron Microscopy. Combining the results obtained from activity analysis, RP-HPLC and CD, we show that the activity recovery was closely related to the conformation of the refolding intermediate, and buffer of very low concentration (e.g. 10mM) alone could efficiently promote correct refolding by maintaining the native-like secondary structure of the intermediate state. This study reveals reasons for lysozyme aggregation and puts new insights into protein and inclusion body refolding.
dc.language.isoen_US
dc.relation<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=15006632&dopt=Abstract">Link to Article in PubMed</a>
dc.relation.urlhttp://dx.doi.org/10.1016/j.biocel.2003.08.015
dc.subjectAmyloid
dc.subjectAnilino Naphthalenesulfonates
dc.subjectChromatography, High Pressure Liquid
dc.subjectCircular Dichroism
dc.subjectGlutathione
dc.subjectHydrogen-Ion Concentration
dc.subjectMuramidase
dc.subjectProtein Binding
dc.subjectProtein Conformation
dc.subjectProtein Folding
dc.subjectProtein Renaturation
dc.subjectSodium Chloride
dc.subjectBiochemistry, Biophysics, and Structural Biology
dc.subjectPharmacology, Toxicology and Environmental Health
dc.titleConformational changes of lysozyme refolding intermediates and implications for aggregation and renaturation
dc.typeJournal Article
dc.source.journaltitleThe international journal of biochemistry and cell biology
dc.source.volume36
dc.source.issue5
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/bmp_pp/116
dc.identifier.contextkey1228628
html.description.abstract<p>It is believed that denatured-reduced lysozyme rapidly forms aggregates during refolding process, which is often worked around by operating at low protein concentrations or in the presence of aggregation inhibitors. However, we found that low concentration buffer alone could efficiently suppress aggregation. Based on this finding, stable equilibrium intermediate states of denatured-reduced lysozyme containing eight free SH groups were obtained in the absence of redox reagents in buffer of low concentrations alone at neutral or mildly alkaline pH. Transition in the secondary structure of the intermediate from native-like to beta-sheet was observed by circular dichroism (CD) as conditions were varied. Dynamic light scattering and ANS-binding studies showed that the self-association accompanied the conformational change and the structure rich in beta-sheet was the intermediate state for aggregation, which could form either amyloid protofibril or amorphous aggregates under different conditions as detected by Electron Microscopy. Combining the results obtained from activity analysis, RP-HPLC and CD, we show that the activity recovery was closely related to the conformation of the refolding intermediate, and buffer of very low concentration (e.g. 10mM) alone could efficiently promote correct refolding by maintaining the native-like secondary structure of the intermediate state. This study reveals reasons for lysozyme aggregation and puts new insights into protein and inclusion body refolding.</p>
dc.identifier.submissionpathbmp_pp/116
dc.contributor.departmentProgram in Molecular Medicine
dc.contributor.departmentDepartment of Biochemistry and Molecular Pharmacology
dc.source.pages795-805


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