Catalytic biomaterials: engineering organophosphate hydrolase to form self-assembling enzymatic hydrogels
Catalytic biomaterials: engineering organophosphate hydrolase to form self-assembling enzymatic hydrogels
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DOI：
10.1093/protein/gzq026
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发表时间：
2010-07-01
影响因子：
2.4
通讯作者：
Banta, Scott
中科院分区：
文献类型：
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作者：
Lu, Hoang D.;Wheeldon, Ian R.;Banta, Scott
有机磷神经毒素污染了环境，每年导致数百万人中毒，并被用作化学武器。结合OP水解酶（OPH）的天然活性的生物材料对于包括OP传感、环境生物修复和预防性去污的应用是感兴趣的。我们已经通过将OPH酶与α-螺旋亮氨酸拉链结构域（H）、非结构化可溶性接头结构域（S）和聚组氨酸纯化标签遗传融合，设计并表征了四种新型水凝胶形成OPH变体。附加的H结构域在酶之间形成物理交联，并使酶能够自组装成水凝胶。添加H和S融合蛋白显著增加了可溶性蛋白的表达水平。具有双末端H结构域的OPH构建体以较低的蛋白质重量比形成水凝胶，并且表现出比用单个H结构域融合修饰的那些变体更高的酶活性。聚组氨酸标签对纯化没有用，但它们不是良性的，因为添加6 His标签增加了蛋白质的水凝胶形成能力，同时降低了k（cat）和K-M值。活性酶水凝胶可由浓缩的未纯化的粗蛋白裂解物制备，显著简化了生物材料的加工和利用。并且，展示了表现出OPH活性的简单蛋白质生物活性表面涂层。水凝胶在长期储存中是稳定的，因为在缓冲液中冷藏5个月后仍保持活性。这些新的蛋白质构建体进一步显示了使用合理的蛋白质设计来创建用于形成催化生物材料的新型双功能自组装单元。
Organophosphate (OP) neurotoxins have contaminated the environment, contributed to millions of poisoning annually, and have been used as chemical weapons. Biomaterials incorporating the native activity of the OP hydrolase (OPH) enzyme are of interest for applications including OP sensing, environmental bioremediation and prophylactic decontamination. We have engineered and characterized four novel hydrogel-forming OPH variants by genetically fusing the OPH enzyme with alpha-helical leucine zipper domains (H), unstructured soluble linker domains (S) and polyhistidine purification tags. The appended H domains form physical cross-links between the enzymes and enable self-assembly of the enzymes into hydrogels. The addition of the H and S fusions significantly increased the expression levels of soluble protein. OPH constructs with biterminal H domains form hydrogels at lower protein weight percents and exhibit higher enzymatic activity than those variants modified with a single H domain fusion. Polyhistidine tags were not useful for purification but they were not benign, as the addition of the 6His tags increased the hydrogel-forming abilities of the proteins with a concomitant reduction in both the k(cat) and K-M values. Active enzymatic hydrogels could be made from concentrated unpurified crude protein lysates, significantly simplifying the processing and utilization of the biomaterials. And, a simple proteinaceous bioactive surface coating exhibiting OPH activity is demonstrated. The hydrogels were stable over long-term storage, as activity was retained after cold storage in buffer after 5 months. These new protein constructs further show the use of rational protein design to create novel, bifunctional, self-assembling units for the formation of catalytic biomaterials.