Division of Bacterial and Rickettsial Diseases, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
Biochem Biophys Res Commun. 2010 Mar 12;393(3):509-13. doi: 10.1016/j.bbrc.2010.02.036. Epub 2010 Feb 10.
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.
衰老标志物蛋白-30（SMP30）已被报道能水解二异丙基氟磷酸酯（DFP），一种化学战剂神经毒剂的替代化合物。因此，SMP30有可能作为一种化学战剂神经毒剂毒性的预防剂使用。我们曾尝试使用多种表达载体在细菌中生成人 SMP30，但得到的都是不溶性和无活性的制剂。在本研究中，我们采用一种联合策略，通过与分子伴侣共表达，在大肠杆菌中生产出正确折叠和具有活性的重组人 SMP30（rHuSMP30）。在 15°C 时与 GroES/GroEL/Tf 共表达，并添加膜流动性调节剂、增加渗透压调节剂，分两步表达，可最大程度提高可溶性和 DFPase 活性。我们的研究结果为探索 rHuSMP30 对抗有机磷和神经毒剂毒性的用途铺平了道路。
