Optimization of human D-amino acid oxidase expression in Escherichia coli.
Human D-amino acid oxidase (hDAAO) is a flavoprotein that plays a key role in the pathophysiology of schizophrenia. So far, the biochemical characterization of this enzyme has been hampered by the difficulty of expressing it in a common heterologous host such as Escherichia coli. Increasing amounts of recombinant hDAAO are indeed required for the investigation of its structure-function relationships and for the screening of new inhibitors to be used in the treatment of schizophrenia. A recombinant hDAAO has been over-expressed in BL21(DE3)Star E. coli cells. By alternating screenings of medium components at flask level and investigating physiological parameters in 2L controlled batch fermentations, an improved, robust and scalable microbial process was set up giving almost a 40- and 4-fold improvement in volumetric productivity and specific activity, respectively. Under these conditions approximately 770 U/L culture hDAAO with a specific activity of approximately 0.4 U/mg protein and a specific productivity of 24.9 U/g biomass were produced. Optimization of medium ingredients, of the time and the amount of inducer's addition, pH control at the moment of induction and harvest, low mechanical shear stress regime during recombinant protein production, represent the factors concurring to achieve the reported expression level. Notably, this expression level is higher than any previously described production of hDAAOs. A yield of 100 mg of pure hDAAO/L culture thus became available in comparison to the 1-10 mg/L previously reported.
Romano
Diego
D
Dipartimento di Biotecnologie e Scienze Molecolari, Università degli Studi dell'Insubria, Varese, Italy.
Molla
Gianluca
G
Pollegioni
Loredano
L
Marinelli
Flavia
F
D003470:Culture Media / Q000737:chemistry / Q000378:metabolism; D003605:D-Amino-Acid Oxidase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005285:Fermentation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D007700:Kinetics; D010865:Pilot Projects; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D012559:Schizophrenia
D003470:Culture Media; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D003605:D-Amino-Acid Oxidase