title: Enhancement of organophosphorus hydrolase yield in Escherichia coli using multiple gene fusions.
abstract: It was previously shown that organophosphorus hydrolase (OPH) expression and purification could be tracked by fluorescence of green fluorescent protein (GFP) when synthesized as an N-terminal fusion with GFP (Cha et al., 2000; Wu et al., 2000). In order to enhance OPH productivity while utilizing the advantage of the reporter protein (GFP), two copies of OPH were cloned in tandem following the gfp(uv) gene (e.g., GFP-OPH(n=2)). Both anti-GFP and anti-OPH Western blots demonstrated that a higher yield was achieved in comparison to the one copy fusion (GFP-OPH). Importantly, the fusion protein was still fluorescent as determined via microscopy. In contrast, a fusion containing two copies of OPH without GFP, and an operon fusion of two OPHs with two independent ribosomal binding sites, did not result in a higher yield than one OPH expressed alone.
authors.affiliation: Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute, College Park, MD 20742, USA.
authors.forename: C F
authors.initials: CF
authors.lastname: Wu
authors.forename: J J
authors.initials: JJ
authors.lastname: Valdes
authors.forename: G
authors.initials: G
authors.lastname: Rao
authors.forename: W E
authors.initials: WE
authors.lastname: Bentley
mesh_terms: D019677:Artificial Gene Fusion; D043303:Aryldialkylphosphatase; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D004950:Esterases / Q000235:genetics*; D018628:Gene Dosage; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D010957:Plasmids
chemical_list: D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004950:Esterases; D043303:Aryldialkylphosphatase