Center of Advanced Studies, Faculty of Military Health Sciences, University of Defence, Hradec Králové, Czech Republic.
Chem Biol Interact. 2010 Sep 6;187(1-3):380-3. doi: 10.1016/j.cbi.2010.03.017. Epub 2010 Mar 15.
Bioscavengers are considered as promising antidotes against organophosphate poisoning. We focused on a bacterial phosphotriesterase (PTE) expressed in Escherichia coli. The main disadvantage of this non-human catalytic bioscavenger is its relatively short half-life in the organism and strong immunogenicity after repeated administration. Therefore, we prepared different methoxy polyethylene glycol (MPEG)-conjugated recombinant PTE as a potential catalytic bioscavenger with the aim to improve its biological properties. Enzyme was modified with two linear monofunctional MPEG derivatives with reactive aldehyde group of molecular weight 2 kDa and 5 kDa. We optimized reaction conditions (reagent ratios, temperature and duration of modification reaction) and we prepared homogeneous population of fully modified recombinant PTE with molecular weight around 52 kDa and 76 kDa, respectively. Modified PTE was characterized using SDS-PAGE and MALDI-TOF and by determining K(m) and V(max). We also investigated thermal stability of modified enzyme at 37 degrees C. Based on our results, for future in vivo evaluation of pharmacokinetics and pharmacodynamics properties, we selected recombinant PTE modified with 5 kDa MPEG aldehyde for its superior thermal stability.
生物清除剂被认为是对抗有机磷中毒的有前途的解毒剂。我们专注于在大肠杆菌中表达的细菌磷酸三酯酶（PTE）。这种非人类催化生物清除剂的主要缺点是其在体内的半衰期相对较短，并且在重复给药后具有很强的免疫原性。因此，我们制备了不同的甲氧基聚乙二醇（MPEG）缀合的重组 PTE 作为一种有潜力的催化生物清除剂，旨在改善其生物学特性。酶用两种具有反应性醛基的线性单官能 MPEG 衍生物修饰，分子量分别为 2 kDa 和 5 kDa。我们优化了反应条件（试剂比、修饰反应温度和时间），并制备了分子量分别约为 52 kDa 和 76 kDa 的均一的完全修饰的重组 PTE 群体。用 SDS-PAGE 和 MALDI-TOF 以及确定 K(m)和 V(max)对修饰的 PTE 进行了表征。我们还研究了修饰酶在 37°C 时的热稳定性。基于我们的结果，为了将来在体内评估药代动力学和药效学特性，我们选择了用 5 kDa MPEG 醛修饰的重组 PTE，因为它具有更好的热稳定性。
