Inclusion body anatomy and functioning of chaperone-mediated in vivo inclusion body disassembly during high-level recombinant protein production in Escherichia coli.

2.50
Hdl Handle:
http://hdl.handle.net/10033/19674
Title:
Inclusion body anatomy and functioning of chaperone-mediated in vivo inclusion body disassembly during high-level recombinant protein production in Escherichia coli.
Authors:
Rinas, Ursula; Hoffmann, Frank; Betiku, Eriola; Estapé, David; Marten, Sabine
Abstract:
During production in recombinant Escherichia coli, the human basic fibroblast growth factor (hFGF-2) partly aggregates into stable cytoplasmic inclusion bodies. These inclusion bodies additionally contain significant amounts of the heat-shock chaperone DnaK, and putative DnaK substrates such as the elongation factor Tu (ET-Tu) and the metabolic enzymes dihydrolipoamide dehydrogenase (LpdA), tryptophanase (TnaA), and d-tagatose-1,6-bisphosphate aldolase (GatY). Guanidinium hydrochloride induced disaggregation studies carried out in vitro on artificial aggregates generated through thermal aggregation of purified hFGF-2 revealed identical disaggregation profiles as hFGF-2 inclusion bodies indicating that the heterogenic composition of inclusion bodies did not influence the strength of interactions of hFGF-2 in aggregates formed in vivo as inclusion bodies compared to those generated in vitro from native and pure hFGF-2 through thermal aggregation. Compared to unfolding of native hFGF-2, higher concentrations of denaturant were required to dissolve hFGF-2 aggregates showing that more energy is required for disruption of interactions in both types of protein aggregates compared to the unfolding of the native protein. In vivo dissolution of hFGF-2 inclusion bodies was studied through coexpression of chaperones of the DnaK and GroEL family and ClpB and combinations thereof. None of the chaperone combinations was able to completely prevent the initial formation of inclusion bodies, but upon prolonged incubation mediated disaggregation of otherwise stable inclusion bodies. The GroEL system was particularly efficient in inclusion body dissolution but did not lead to a corresponding increase in soluble hFGF-2 rather was promoting the proteolysis of the recombinant growth factor. Coproduction of the disaggregating DnaK system and ClpB in conjunction with small amounts of the chaperonins GroELS was most efficient in disaggregation with concomitant formation of soluble hFGF-2. Thus, fine-balanced coproduction of chaperone combinations can play an important role in the production of soluble recombinant proteins with a high aggregation propensity not through prevention of aggregation but predominantly through their disaggregating properties.
Affiliation:
Biochemical Engineering Division, GBF German Research Center for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. URI@gbf.de
Citation:
Inclusion body anatomy and functioning of chaperone-mediated in vivo inclusion body disassembly during high-level recombinant protein production in Escherichia coli. 2007, 127 (2):244-57 J. Biotechnol.
Journal:
Journal of biotechnology
Issue Date:
1-Jan-2007
URI:
http://hdl.handle.net/10033/19674
DOI:
10.1016/j.jbiotec.2006.07.004
PubMed ID:
16945443
Type:
Article
Language:
en
ISSN:
0168-1656
Appears in Collections:
Publications from Division of Molekulare Struktur Biologie (MOSB)

Full metadata record

DC FieldValue Language
dc.contributor.authorRinas, Ursula-
dc.contributor.authorHoffmann, Frank-
dc.contributor.authorBetiku, Eriola-
dc.contributor.authorEstapé, David-
dc.contributor.authorMarten, Sabine-
dc.date.accessioned2008-03-04T14:38:17Z-
dc.date.available2008-03-04T14:38:17Z-
dc.date.issued2007-01-01-
dc.identifier.citationInclusion body anatomy and functioning of chaperone-mediated in vivo inclusion body disassembly during high-level recombinant protein production in Escherichia coli. 2007, 127 (2):244-57 J. Biotechnol.en
dc.identifier.issn0168-1656-
dc.identifier.pmid16945443-
dc.identifier.doi10.1016/j.jbiotec.2006.07.004-
dc.identifier.urihttp://hdl.handle.net/10033/19674-
dc.description.abstractDuring production in recombinant Escherichia coli, the human basic fibroblast growth factor (hFGF-2) partly aggregates into stable cytoplasmic inclusion bodies. These inclusion bodies additionally contain significant amounts of the heat-shock chaperone DnaK, and putative DnaK substrates such as the elongation factor Tu (ET-Tu) and the metabolic enzymes dihydrolipoamide dehydrogenase (LpdA), tryptophanase (TnaA), and d-tagatose-1,6-bisphosphate aldolase (GatY). Guanidinium hydrochloride induced disaggregation studies carried out in vitro on artificial aggregates generated through thermal aggregation of purified hFGF-2 revealed identical disaggregation profiles as hFGF-2 inclusion bodies indicating that the heterogenic composition of inclusion bodies did not influence the strength of interactions of hFGF-2 in aggregates formed in vivo as inclusion bodies compared to those generated in vitro from native and pure hFGF-2 through thermal aggregation. Compared to unfolding of native hFGF-2, higher concentrations of denaturant were required to dissolve hFGF-2 aggregates showing that more energy is required for disruption of interactions in both types of protein aggregates compared to the unfolding of the native protein. In vivo dissolution of hFGF-2 inclusion bodies was studied through coexpression of chaperones of the DnaK and GroEL family and ClpB and combinations thereof. None of the chaperone combinations was able to completely prevent the initial formation of inclusion bodies, but upon prolonged incubation mediated disaggregation of otherwise stable inclusion bodies. The GroEL system was particularly efficient in inclusion body dissolution but did not lead to a corresponding increase in soluble hFGF-2 rather was promoting the proteolysis of the recombinant growth factor. Coproduction of the disaggregating DnaK system and ClpB in conjunction with small amounts of the chaperonins GroELS was most efficient in disaggregation with concomitant formation of soluble hFGF-2. Thus, fine-balanced coproduction of chaperone combinations can play an important role in the production of soluble recombinant proteins with a high aggregation propensity not through prevention of aggregation but predominantly through their disaggregating properties.en
dc.language.isoenen
dc.subject.meshEscherichia colien
dc.subject.meshEscherichia coli Proteinsen
dc.subject.meshFibroblast Growth Factor 2en
dc.subject.meshGroEL Proteinen
dc.subject.meshGroES Proteinen
dc.subject.meshHSP40 Heat-Shock Proteinsen
dc.subject.meshHSP70 Heat-Shock Proteinsen
dc.subject.meshHumansen
dc.subject.meshInclusion Bodiesen
dc.subject.meshMolecular Chaperonesen
dc.subject.meshMutationen
dc.subject.meshProtein Denaturationen
dc.subject.meshProtein Foldingen
dc.subject.meshProtein Structure, Quaternaryen
dc.subject.meshProteomeen
dc.subject.meshRecombinant Proteinsen
dc.subject.meshSolubilityen
dc.subject.meshThermodynamicsen
dc.titleInclusion body anatomy and functioning of chaperone-mediated in vivo inclusion body disassembly during high-level recombinant protein production in Escherichia coli.en
dc.typeArticleen
dc.contributor.departmentBiochemical Engineering Division, GBF German Research Center for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. URI@gbf.deen
dc.identifier.journalJournal of biotechnologyen
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