title: Efficient solubilization of inclusion bodies.
abstract: The overexpression of recombinant proteins in Escherichia coli leads in most cases to their accumulation in the form of insoluble aggregates referred to as inclusion bodies (IBs). To obtain an active product, the IBs must be solubilized and thereafter the soluble monomeric protein needs to be refolded. In this work we studied the solubilization behavior of a model-protein expressed as IBs at high protein concentrations, using a statistically designed experiment to determine which of the process parameters, or their interaction, have the greatest impact on the amount of soluble protein and the fraction of soluble monomer. The experimental methodology employed pointed out an optimum balance between maximum protein solubility and minimum fraction of soluble aggregates. The optimized conditions solubilized the IBs without the formation of insoluble aggregates; moreover, the fraction of soluble monomer was approximately 75% while the fraction of soluble aggregates was approximately 5%. Overall this approach guarantees a better use of the solubilization reagents, which brings an economical and technical benefit, at both large and lab scale and may be broadly applicable for the production of recombinant proteins.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.
authors.forename: Esteban J
authors.initials: EJ
authors.lastname: Freydell
authors.forename: Marcel
authors.initials: M
authors.lastname: Ottens
authors.forename: Michel
authors.initials: M
authors.lastname: Eppink
authors.forename: Gijs
authors.initials: G
authors.lastname: van Dedem
authors.forename: Luuk
authors.initials: L
authors.lastname: van der Wielen
mesh_terms: D005591:Chemical Fractionation / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002479:Inclusion Bodies / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012995:Solubility
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins