Abstract
Soluble protein fractions are often considered containing exclusively monodispersed and correctly folded molecules. This is not the case, being soluble aggregates of different complexity widely represented in such fractions. The use of fusing target protein domains to highly soluble carriers may strongly contribute to soluble aggregate accumulation. Therefore, reliable analytical methods must be used to evaluate the biophysical characteristics of soluble proteins. On the other hand, conventional methodologies are often technically demanding and time consuming. In this method paper, a protocol is presented that enables the rapid evaluation of the protein monodispersity from the initial step aimed at screening several conditions in parallel to the setup of the complete protocol for large-scale purification. The analysis is performed by means of simple lab equipment and starting from small sample volumes.
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References
Sachdev, D., Chirgwin, J.M. (1999) Properties of soluble fusions between mammalian aspartic proteases and bacterial maltose-binding protein. Biochem. J. 338, 77–81.
Nominé, Y., Ristriani, T., Laurent, C., et al., (2001) A strategy for optimizing the monodispersity of fusion proteins: application to purification of recombinant HPV E6 oncoprotein. Prot. Engineer. 14, 297–305.
Schrödel, A., de Marco, A. (2005) Characterization of the aggregates formed during recombinant protein expression in bacteria. BMC Biochem. 6, 10.
Martinez-Alonso, M., Gonzalez-Montalban, N., Garcia-Fruitos, E., et al., (2008) The functional quality of soluble recombinant polypetides produced in Escherichia coli is defined by a wide conformational spectrum. Appl. Environ. Microbiol. 74, 7431–7433.
Martinez-Alonso, M., Garcia-Fruitos, E., Villaverde, A. (2008) Yield, solubility and conformational quality of soluble proteins are not simultaneously favored in recombinant Escherichia coli. Biotechnol Bioeng. 101, 1353–1358.
Nataliello, A., Santarella, R., Doglia, S.M., et al., (2008) Physical and chemical perturbations induce the formation of protein aggregates with different structural features. Protein Expr. Purif. 58, 356–361.
Nataliello, A., Liu, J., Ami, D., et al., (2008) The osmolyte betaine promotes protein misfolding and disruption of protein aggregates. Proteins 75, 509–517.
Sala, E., de Marco, A. (2010) Screening optimized protein purification protocols by coupling small-scale expression and mini-size exclusion chromatography. Prot. Expr. Purif. 74, 231–235.
Capasso, P., Aliprandi, M., Ossolengo, G., et al., (2009) Monodispersity of recombinant Cre recombinase correlates with its effectiveness in vivo. BMC Biotechnol. 9, 80.
Berger, I., Fitzgerald, D.J., Richmond, T.J. (2004) Baculovirus expression system for heterologous multiprotein complexes. Nat. Biotechnol. 22, 1583–1587.
de Marco, A. (2007) Protocol for preparing proteins with improved solubility by co-expression with molecular chaperones in Escherichia coli. Nature Protocols 2, 2632–2639.
Wen, J., Arakawa, T., Philo, J.S. (2004) Size-exclusion chromatography with on-line light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions. Anal. Biochem. 240, 155–166.
Liu, S., Tobias, R., McClure, S., et al., (1997) Removal of endotoxin from recombinant protein preparations. Clinical Biochem. 30, 455–463.
Roberts, M.F. (2005) Organic compatible solutes of halotolerant and halophilic microorganisms. Saline Sys. 1, 5.
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de Marco, A. (2012). Optimization of Purification Protocols Based on the Step-by-Step Monitoring of the Protein Aggregates in Soluble Fractions. In: Lorence, A. (eds) Recombinant Gene Expression. Methods in Molecular Biology, vol 824. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-433-9_7
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DOI: https://doi.org/10.1007/978-1-61779-433-9_7
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-61779-433-9
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