title: Increasing the catalytic activity of Bilirubin oxidase from Bacillus pumilus: Importance of host strain and chaperones proteins.
abstract: Aggregation of recombinant proteins into inclusion bodies (IBs) is the main problem of the expression of multicopper oxidase in Escherichia coli. It is usually attributed to inefficient folding of proteins due to the lack of copper and/or unavailability of chaperone proteins. The general strategies reported to overcome this issue have been focused on increasing the intracellular copper concentration. Here we report a complementary method to optimize the expression in E. coli of a promising Bilirubin oxidase (BOD) isolated from Bacillus pumilus. First, as this BOD has a disulfide bridge, we switched E.coli strain from BL21 (DE3) to Origami B (DE3), known to promote the formation of disulfide bridges in the bacterial cytoplasm. In a second step, we investigate the effect of co-expression of chaperone proteins on the protein production and specific activity. Our strategy allowed increasing the final amount of enzyme by 858% and its catalytic rate constant by 83%.
authors.affiliation: CNRS, CRPP, UPR 8641, F-33600 Pessac, France; Univ. Bordeaux, CRPP, UPR 8641, F-33600 Pessac, France.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Gounel
authors.affiliation: CNRS, CRPP, UPR 8641, F-33600 Pessac, France; Univ. Bordeaux, CRPP, UPR 8641, F-33600 Pessac, France.
authors.forename: Jad
authors.initials: J
authors.lastname: Rouhana
authors.affiliation: CNRS, CRPP, UPR 8641, F-33600 Pessac, France; Univ. Bordeaux, CRPP, UPR 8641, F-33600 Pessac, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Stines-Chaumeil
authors.affiliation: CNRS, CRPP, UPR 8641, F-33600 Pessac, France; Univ. Bordeaux, CRPP, UPR 8641, F-33600 Pessac, France.
authors.forename: Marine
authors.initials: M
authors.lastname: Cadet
authors.affiliation: CNRS, CRPP, UPR 8641, F-33600 Pessac, France; Univ. Bordeaux, CRPP, UPR 8641, F-33600 Pessac, France. Electronic address: mano@crpp-bordeaux.cnrs.fr.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Mano
mesh_terms: D000069979:Bacillus pumilus / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D044925:Oxidoreductases Acting on CH-CH Group Donors; C042401:bilirubin oxidase
keywords: Bacterial laccase; Bilirubin oxidase; Chaperone proteins; Escherichia coli; Heterologous expression