Inclusion body anatomy and functioning of chaperone-mediated in vivo inclusion body disassembly during high-level recombinant protein production in Escherichia coli.
Name:
Publisher version
View Source
Access full-text PDFOpen Access
View Source
Check access options
Check access options
Average rating
Cast your vote
You can rate an item by clicking the amount of stars they wish to award to this item.
When enough users have cast their vote on this item, the average rating will also be shown.
Star rating
Your vote was cast
Thank you for your feedback
Thank you for your feedback
Issue Date
2007-01-01
Metadata
Show full item recordAbstract
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.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.Affiliation
Biochemical Engineering Division, GBF German Research Center for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. URI@gbf.deJournal
Journal of biotechnologyPubMed ID
16945443Type
ArticleLanguage
enISSN
0168-1656ae974a485f413a2113503eed53cd6c53
10.1016/j.jbiotec.2006.07.004
Scopus Count
The following license files are associated with this item:
Related articles
- Kinetics of heat-shock response and inclusion body formation during temperature-induced production of basic fibroblast growth factor in high-cell-density cultures of recombinant Escherichia coli.
- Authors: Hoffmann F, Rinas U
- Issue date: 2000 Nov-Dec
- DnaK and DnaJ facilitated the folding process and reduced inclusion body formation of magnesium transporter CorA overexpressed in Escherichia coli.
- Authors: Chen Y, Song J, Sui SF, Wang DN
- Issue date: 2003 Dec
- Bacterial inclusion bodies are cytotoxic in vivo in absence of functional chaperones DnaK or GroEL.
- Authors: González-Montalbán N, Carrió MM, Cuatrecasas S, Arís A, Villaverde A
- Issue date: 2005 Sep 10
- Consortium of fold-catalyzing proteins increases soluble expression of cyclohexanone monooxygenase in recombinant Escherichia coli.
- Authors: Lee DH, Kim MD, Lee WH, Kweon DH, Seo JH
- Issue date: 2004 Feb
- Divergent genetic control of protein solubility and conformational quality in Escherichia coli.
- Authors: García-Fruitós E, Martínez-Alonso M, Gonzàlez-Montalbán N, Valli M, Mattanovich D, Villaverde A
- Issue date: 2007 Nov 16