Inclusion body anatomy and functioning of chaperone-mediated in vivo inclusion body disassembly during high-level recombinant protein production in Escherichia coli
| Autor: | Sabine Marten, Ursula Rinas, Frank Hoffmann, David Estapé, Eriola Betiku |
|---|---|
| Přispěvatelé: | Biochemical Engineering Division, GBF German Research Center for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. URI@gbf.de |
| Rok vydání: | 2007 |
| Předmět: |
Protein Denaturation
Protein Folding Proteome GroES Protein Bioengineering Protein aggregation Applied Microbiology and Biotechnology Inclusion bodies Chaperonin Chaperonin 10 Escherichia coli Humans HSP70 Heat-Shock Proteins Protein Structure Quaternary Inclusion Bodies biology Escherichia coli Proteins Chaperonin 60 General Medicine HSP40 Heat-Shock Proteins GroEL Protein GroEL Recombinant Proteins Recombinant Growth Factor Solubility Biochemistry Chaperone (protein) Mutation biology.protein Thermodynamics Fibroblast Growth Factor 2 Protein folding CLPB Molecular Chaperones Biotechnology |
| Zdroj: | Journal of Biotechnology. 127:244-257 |
| ISSN: | 0168-1656 |
| DOI: | 10.1016/j.jbiotec.2006.07.004 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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