Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 393, Issue 3, Pages 509-513Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2010.02.036
Keywords
Senescence marker protein-30 (SMP30); Diisopropyl fluorophosphatase (DFPase); Bacterial expression; Molecular chaperones; Solubility; Two-step growth
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Funding
- Defense Threat Reduction Agency [D.0024_07_WR_C]
- Department of Defense (DOD)
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Senescence marker protein-30 (SMP30) has been reported to hydrolyze diisopropyl fluorophosphate (DFP), a surrogate compound of chemical warfare nerve agents. Thus, SMP30 has the potential to be useful as a prophylactic against chemical warfare nerve agent toxicity. Our efforts to generate human SMP30 in bacteria using a variety of expression vectors invariably resulted in insoluble and inactive preparations. In this study, properly folded and active recombinant human SMP30 (rHuSMP30) was produced in Escherichia coli by coexpressing it with molecular chaperones in a combined strategy. The coexpression of rHuSMP30 with GroES/GroEL/Tf at 15 degrees C, combined with the addition of a membrane fluidizer, increased osmolytes, and a two-step expression resulted in the highest enhancement of solubility and DFPase activity. Our results pave the way for exploring the use of rHuSMP30 against organophosphate and nerve agent toxicity. (C) 2010 Elsevier Inc. All rights reserved.
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