Abstract
Owing to simplicity, speed, cost advantage, and a generally high product yield, expression in Escherichia coli is the method of choice for the production of large amounts of protein. However, because of the high expression level, proteins often accumulate within the cells as insoluble aggregates called inclusion bodies. The inclusion body protein is misfolded and biologically inactive and, thus, needs to be refolded into its native conformation. There is no universal method for refolding inclusion bodies and optimal conditions have to be determined empirically for any given protein. Here, we describe a simple and efficient refolding protocol for the catalytic domain of type 4 cyclic nucleotide phosphodiesterases (PDE4s). This method has the potential for adaptation to other PDE subtypes.
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References
Chalmers, J. J., Kim, E., Telford, J. N., Wong, E. Y., Tacon, W. C., Shuler, M. L., and Wilson, D. B. (1990) Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor. Appl. Environ. Microbiol. 56, 104–111.
Kopetzki, E., Schumacher, G., and Buckel, P. (1989) Control of formation of active soluble or inactive insoluble baker’s yeast alpha-glucosidase PI in Escherichia coli by induction and growth conditions. Mol. Gen. Genet. 216, 149–155.
Kapust, R. B. and Waugh, D. S. (1999) Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci. 8, 1668–1674.
Kovala, T., Sanwal, B. D., and Ball, E. H. (1997) Recombinant expression of a type IV, cAMP-specific phosphodiesterase: characterization and structure-function studies of deletion mutants. Biochemistry 36, 2968–2976.
Gatenby, A. A., Viitanen, P. V., and Lorimer, G. H. (1990) Chaperonin assisted polypeptide folding and assembly: implications for the production of functional proteins in bacteria. Trends Biotechnol. 8, 354–358.
Machida, S., Yu, Y., Singh, S. P., Kim, J. D., Hayashi, K., and Kawata, Y. (1998) Overproduction of beta-glucosidase in active form by an Escherichia coli system coexpressing the chaperonin GroEL/ES. FEMS Microbiol. Lett. 159, 41–46.
Makrides, S. C. (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Rev. 60, 512–538.
Rudolph, R., Böhm, G., Lilie, H., and Jaenicke, R. (1997) Folding proteins, in Protein Function, A Practical Approach (Creighton, T. E., ed.), IRL, Oxford, UK, pp. 57–99.
Rudolph, R. and Lilie, H. (1996) In vitro folding of inclusion body proteins. FASEB J. 10, 49–56.
Tsumoto, K., Ejima, D., Kumagai, I., and Arakawa, T. (2003) Practical considerations in refolding proteins from inclusion bodies. Protein Expr. Purif. 28, 1–8.
Lilie, H., Schwarz, E., and Rudolph, R. (1998) Advances in refolding of proteins produced in E. coli. Curr. Opin. Biotechnol. 9, 497–501.
Middelberg, A. P. (2002) Preparative protein refolding. Trends Biotechnol. 20, 437–443.
Guise, A. D., West, S. M., and Chaudhuri, J. B. (1996) Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies. Mol. Biotechnol. 6, 53–64.
Chaudhuri, J. B. (1994) Refolding recombinant proteins: process strategies and novel approaches. Ann. NY Acad. Sci. 721, 374–385.
Francis, S. H., Turko, I. V., and Corbin, J. D. (2001) Cyclic nucleotide phosphodiesterases: relating structure and function. Prog. Nucleic Acid Res. Mol. Biol. 65, 1–52.
Xu, R. X., Hassell, A. M., Vanderwall, D., et al. (2000) Atomic structure of PDE4: insights into phosphodiesterase mechanism and specificity. Science 288, 1822–1825.
Richter, W., Hermsdorf, T., Lilie, H., Egerland, U., Rudolph, R., Kronbach, T., and Dettmer, D. (2000) Refolding, purification, and characterization of human recombinant PDE4A constructs expressed in Escherichia coli. Protein Expr. Purif. 19, 375–383.
Richter, W., Hermsdorf, T., Kronbach, T., and Dettmer, D. (2002) Refolding and purification of recombinant human PDE7A expressed in Escherichia coli as inclusion bodies. Protein Expr. Purif. 25, 138–148.
Armstrong, N., de Lencastre, A., and Gouaux, E. (1999) A new protein folding screen: application to the ligand binding domains of a glutamate and kainate receptor and to lysozyme and carbonic anhydrase. Protein Sci. 8, 1475–1483.
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We are indebted to Caren Spencer for editorial work on the manuscript.
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Richter, W., Hermsdorf, T., Dettmer, D. (2005). Renaturation of the Catalytic Domain of PDE4A Expressed in Escherichia coli as Inclusion Bodies. In: Lugnier, C. (eds) Phosphodiesterase Methods and Protocols. Methods In Molecular Biology™, vol 307. Humana Press. https://doi.org/10.1385/1-59259-839-0:155
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DOI: https://doi.org/10.1385/1-59259-839-0:155
Publisher Name: Humana Press
Print ISBN: 978-1-58829-314-5
Online ISBN: 978-1-59259-839-7
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