Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
J Microbiol Biotechnol. 2012 Feb;22(2):234-8. doi: 10.4014/jmb.1104.04011.
Recombinant Escherichia coli displaying organophosphorus hydrolase (OPH) was used to overcome the diffusion barrier limitation of organophosphorus pesticides. A new anchor system derived from the N-terminal domain of ice-nucleation protein from Pseudomonas syringae InaV (InaV-N) was used to display OPH onto the surface. The designed sequence was cloned in the vector pET-28a(+) and then was expressed in E. coli. Tracing of the expression location of the recombinant protein using SDS-PAGE showed the presentation of OPH by InaV-N on the outer membrane, and the ability of recombinant E. coli to utilize diazinon as the sole source of energy, without growth inhibition, indicated its significant activity. The location of OPH was detected by comparing the activity of the outer membrane fraction with the inner membrane and cytoplasm fractions. Studies revealed that recombinant E. coli can degrade 50% of 2 mM chlorpyrifos in 2 min. It can be concluded that InaV-N can be used efficiently to display foreign functional protein, and these results highlight the high potential of an engineered bacterium to be used in bioremediation of pesticide-contaminated sources in the environment.
重组大肠杆菌展示有机磷水解酶（OPH）被用于克服有机磷农药的扩散障碍限制。一种新的锚定系统源自丁香假单胞菌 InaV 的 N 端结构域（InaV-N），用于将 OPH 展示在表面上。设计的序列被克隆到载体 pET-28a(+) 中，然后在大肠杆菌中表达。使用 SDS-PAGE 追踪重组蛋白的表达位置表明，InaV-N 将 OPH 呈现在外膜上，而重组大肠杆菌能够利用敌百虫作为唯一的能量来源，而没有生长抑制，表明其具有显著的活性。通过比较外膜部分、内膜部分和细胞质部分的活性来检测 OPH 的位置。研究表明，重组大肠杆菌可以在 2 分钟内降解 2mM 毒死蜱的 50%。可以得出结论，InaV-N 可以有效地用于展示外源功能蛋白，这些结果突出了工程菌在环境中用于农药污染源生物修复的巨大潜力。
