A green fluorescent protein fusion strategy for monitoring the expression, cellular location, and separation of biologically active organophosphorus hydrolase |
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| Authors: | C-F Wu H J Cha G Rao J J Valdes W E Bentley |
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| Institution: | (1) Center for Agricultural Biotechnology, University of Maryland, Biotechnology Institute, University of Maryland, College Park, MD 20742, USA e-mail: bentley@eng.umd.edu Tel.: +1-301-4054321 Fax: +1-301-3149075, US;(2) Medical Biotechnology Center, University of Maryland Biotechnology Institute, University of Maryland, College Park, MD 20742, USA, US;(3) Department of Chemical Engineering, University of Maryland, College Park, MD 20742, USA, US;(4) Department of Chemical and Biochemical Engineering, University of Maryland, Baltimore, MD 21250, USA, TP;(5) U.S. Army ERDEC, Aberdeen Proving Ground, MD 21010, USA, US |
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| Abstract: | Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. We have developed a versatile
monitoring technique for detecting the amount of OPH during the expression and purification steps. This involves fusion of
the gene for green fluorescent protein (GFP) to the 5′ end of the OPH gene and subsequent expression in Escherichia coli. The synthesized fusion protein was directly visualized due to the optical properties of GFP. Western blot analyses showed
that the correct fusion protein was expressed after IPTG-induction. Also, the in vivo GFP fluorescence intensity was proportional
to the OPH enzyme activity. Moreover, the OPH, which forms a dimer in its active state, retained activity while fused to GFP.
Enterokinase digestion experiments showed that OPH was separated from the GFP reporter after purification via immobilized
metal affinity chromatography, which in turn was monitored by fluorescence. The strategy of linking GFP to OPH has enormous
potential for improving enzyme production efficiency, as well as enhancing field use, as it can be monitored at low concentrations
with inexpensive instrumentation based on detecting green fluorescence.
Received: 27 April 1999 / Received last revision: 18 October 1999 / Accepted: 1 November 1999 |
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