Thermally triggered purification and immobilization of elastin-OPH fusions.
A bifunctional fusion protein consisting of organophosphorus hydrolase (OPH) and elastin-like polypeptide (ELP) was synthesized for the detoxification of organophosphorus compounds. ELPs undergo a reversible phase transition upon an increase in temperature, forming hydrophobic aggregates. This thermally triggered property of phase transition allows for a simple and rapid means of purifying the fusion protein. Over 1,300-fold purification was achieved after only 2 cycles of inverse phase transition. The purified fusion protein showed identical kinetic properties as the native OPH with only a modest 10% increase in K(m) and a 5% decrease of K(cat). The ability of the ELP domain to form collapsed aggregates also improved long-term stability of the fusion enzyme. Aggregated ELP-OPH retained nearly 100% activity over a span of three weeks. In addition to facilitating purification and stability, the ELP moiety served as a hydrophobic tag for one-step immobilization of the fusion protein onto hydrophobic surfaces. The ELP-OPH was capable of rapidly degrading paraoxon while immobilized. The protein also retained ELP functionality of reversible phase transition thereby allowing for the regeneration of the treated surface. This technology offers a swift and convenient means for purification, immobilization, and regeneration of OPH onto a variety of hydrophobic surfaces by simple environmental triggers.
Shimazu
Mark
M
Environmental Toxicology Program, University of California, Riverside 92521, USA.
Mulchandani
Ashok
A
Chen
Wilfred
W
D043303:Aryldialkylphosphatase; D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D004549:Elastin / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D004549:Elastin; D004950:Esterases; D043303:Aryldialkylphosphatase