Abstract
The expression of recombinant proteins as insoluble inclusion bodies (IB) has the advantage to separate insoluble aggregates from soluble bacterial molecules, thus obtaining proteins with a high degree of purity. Even aggregated, the proteins in IB often present native-like secondary and tertiary structures, which can be maintained as long as solubilization is carried out in non-denaturing condition. High pressure solubilizes IB by weakening hydrophobic interactions, while alkaline pH solubilizes aggregates by electrostatic repulsion. The combination of high pressure and alkaline pH is effective for IB solubilization at a mild, non-denaturing condition, which is useful for subsequent refolding. Here, we describe the expression of recombinant proteins in Escherichia coli using a rich medium to obtain high expression levels, bacterial lysis, and washing of the IB to obtain products of high purity, and, finally, the solubilization and high yield of refolded proteins using high pressure and alkaline pH.
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References
Ami D, Natalello A, Taylor G, Tonon G, Maria Doglia S (2006) Structural analysis of protein inclusion bodies by Fourier transform infrared microspectroscopy. Biochim Biophys Acta 1764:793–799. https://doi.org/10.1016/j.bbapap.2005.12.005
Chura-Chambi RM, Prieto-da-Silva ARB, Di Lela MM, Oliveira JE et al (2022) High level SARS-CoV-2 nucleocapsid refolding using mild condition for inclusion bodies solubilization: application of high pressure at pH 9.0. PLoS One 17:e0262591. https://doi.org/10.1371/journal.pone.026259.1
Chura-Chambi RM, Farah SC, Morganti L (2022) Human growth hormone inclusion bodies present native-like secondary and tertiary structures which can be preserved by mild solubilization for refolding, vol 21. Microbiol Cell Factories, p 164. https://doi.org/10.1186/s12934-022-01887-1
Rathore AS, Bade P, Joshi V, Pathak M, Pattanayek SK (2013) Refolding of biotech therapeutic proteins expressed in bacteria: review. J Chem Technol Biotechnol 88:1794–1806. https://doi.org/10.1002/jctb.4152
Crisman RI, Randolph TW (2009) Refolding of proteins from inclusion bodies is favored by a diminished hydrophobic effect at elevated pressures. Biotechnol Bioeng 102:483–492. https://doi.org/10.1002/bit.22082
St John RJ, Carpenter JF, Randolph TW (1999) High pressure fosters protein refolding from aggregates at high concentrations. Proc Natl Acad Sci U S A 96:13029–13033. https://doi.org/10.1073/pnas.96.23.13029
Chura-Chambi RM, Genova LA, Affonso R, Morganti L (2008) Refolding of endostatin from inclusion bodies using high hydrostatic pressure. Anal Biochem 379:32–39. https://doi.org/10.1016/j.ab.2008.04.024
St John RJ, Carpenter JF, Balny C, Randolph TW (2001) High pressure refolding of recombinant human growth hormone from insoluble aggregates. Structural transformations, kinetic barriers, and energetics. J Biol Chem 276:46856–46863. https://doi.org/10.1074/jbc.M107671200
Yongsawatdigul J, Park JW (2004) Effects of alkali and acid solubilization on gelation characteristics of rockfish muscle proteins. J Food Sci 69:C499–C505. https://doi.org/10.1111/j.1365-2621.2004.tb13642.x
Patra AK, Mukhopadhyay R, Mukhija R, Krishnan A et al (2000) Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli. Protein Expr Purif 18:182–192. https://doi.org/10.1006/prep.1999.1179
Chura-Chambi RM, da Silva CMR, Pereira LR, Bartolini P et al (2019) Protein refolding based on high hydrostatic pressure and alkaline pH: application on a recombinant dengue virus NS1 protein. PLoS One 14:e0211162. https://doi.org/10.1371/journal.pone.0211162
da Silva CMR, Chura-Chambi RM, Pereira LR, Cordeiro Y et al (2018) Association of high pressure and alkaline condition for solubilization of inclusion bodies and refolding of the NS1 protein from zika virus. BMC Biotechnol 18:78. https://doi.org/10.1186/s12896-018-0486-2
Jensen EB, Carlsen S (1990) Production of recombinant human growth-hormone in Escherichia-Coli – expression of different precursors and physiological-effects of glucose, acetate, and salts. Biotechnol Bioeng 36:1–11. https://doi.org/10.1002/bit.260360102
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. https://doi.org/10.1038/227680a0
Qoronfleh MW, Hesterberg LK, Seefeldt MB (2007) Confronting high-throughput protein refolding using high pressure and solution screens. Protein Expr Purif 55:209–224. https://doi.org/10.1016/j.pep.2007.05.014
Malavasi NV, Foguel D, Bonafe CFS, Braga CACA et al (2011) Protein refolding at high pressure: optimization using eGFP as a model. Process Biochem 46:512–518. https://doi.org/10.1016/j.procbio.2010.10.002
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Morganti, L., Chura-Chambi, R.M. (2023). Using High Pressure and Alkaline pH for Refolding. In: Kopp, J., Spadiut, O. (eds) Inclusion Bodies. Methods in Molecular Biology, vol 2617. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2930-7_12
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DOI: https://doi.org/10.1007/978-1-0716-2930-7_12
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