title: Effect of controlled redox potential and dissolved oxygen on the in vitro refolding of an E. coli alkaline phosphatase and chicken lysozyme.
abstract: The development of efficient purification strategies of recombinant active protein derived from inclusion bodies requires the knowledge of the effect of environmental variables, such as redox potential (RP) and dissolved oxygen tension (DOT), in order to control the protein folding process. However, that information is scarce and only few in vitro studies of the impact of such variables have been reported under constant controlled conditions. In this work, the effect of controlled RP and DOT on the refolding of E. coli alkaline phosphatase (AP) and chicken lysozyme (CL) enzymes were studied. Disulphide bonds of both enzymes were reduced in an instrumented vessel using 2-mercaptoethanol and nitrogen. In the latter case, guanidine hydrochloride was also used to denature the protein. Such conditions caused protein conformational changes, as determined by the intrinsic fluorescence spectra that correlated with a decrease on the activity in both cases. Reduced enzymes were then oxidized, under different constant and predetermined RP or DOT, by manipulating the gas composition in the vessel. Folding kinetics were followed as the recovery of enzyme activity. Results showed that the percentage of recovery and rate of increase of enzymatic activity directly depended on the RP and DOT. A higher folding efficiency was found under controlled DOT compared to controlled RP conditions. These results are useful for establishing protein folding strategies to improve the recovery of active protein from inclusion bodies.
authors.affiliation: Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Av. Universidad 1010, Col. Chamilpa, Cuernavaca, Morelos CP 62100, Mexico.
authors.forename: Angélica
authors.initials: A
authors.lastname: Meneses-Acosta
authors.forename: Luis Rodolfo
authors.initials: LR
authors.lastname: Vizcaíno-Meza
authors.forename: Hector G
authors.initials: HG
authors.lastname: Ayala-Castro
authors.forename: Martha A
authors.initials: MA
authors.lastname: Contreras
authors.forename: Jaime
authors.initials: J
authors.lastname: Ortega-López
authors.forename: Octavio T
authors.initials: OT
authors.lastname: Ramírez
mesh_terms: D000469:Alkaline Phosphatase / Q000187:drug effects*; D000818:Animals; D002645:Chickens / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D009113:Muramidase / Q000187:drug effects*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000032:analysis / Q000494:pharmacology*; D011489:Protein Denaturation; D017510:Protein Folding / Q000187:drug effects*; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D000469:Alkaline Phosphatase; D009113:Muramidase; D010100:Oxygen