Enhancement of organophosphorus hydrolase yield in Escherichia coli using multiple gene fusions.
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.
Wu
C F
CF
Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute, College Park, MD 20742, USA.
Valdes
J J
JJ
Rao
G
G
Bentley
W E
WE
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
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004950:Esterases; D043303:Aryldialkylphosphatase