Osmolyte-mediated encapsulation of proteins inside MS2 viral capsids.
Osmolyte-mediated encapsulation of proteins inside MS2 viral capsids.
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DOI：
10.1021/nn302183h
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发表时间：
2012-10-23
期刊：
影响因子：
17.1
通讯作者：
Tullman-Ercek, Danielle
中科院分区：
文献类型：
--
作者：
Glasgow, Jeff E.;Capehart, Stacy L.;Francis, Matthew B.;Tullman-Ercek, Danielle
将酶封装在纳米尺寸的隔室中具有在体内和体外增强和控制酶活性的潜力。尽管有这种潜力，但在不同条件下，在明确定义的隔室中封装的效果的定量数据很少。为了更深入地了解这些影响，我们已经表征了两种用于将异源分子封装在噬菌体MS 2病毒衣壳内的改进方法。首先，将DNA寡聚体连接到感兴趣的分子上，并将其与MS 2外壳蛋白二聚体一起孵育，产生重新组装的衣壳，其包装标记的分子。添加蛋白质稳定渗透剂，三甲基胺-N-氧化物（TMAO），显着增加重组的产量。其次，我们发现，表达的蛋白质与遗传编码的带负电荷的肽标签也可以诱导衣壳重组，导致高产量的重组衣壳含有蛋白质。第二种方法用于包封用16个氨基酸肽标记的碱性磷酸酶。发现纯化的包封酶具有相同的Km值和比游离酶略低的kcat值，表明这种包封方法对酶动力学的影响最小。这种方法提供了一种实用的和潜在的可扩展的方式来研究在基于蛋白质的隔室中包封酶的复杂效应。
The encapsulation of enzymes in nanometer-sized compartments has the potential to enhance and control enzymatic activity, both in vivo and in vitro. Despite this potential, there are little quantitative data on the effect of encapsulation in a well-defined compartment under varying conditions. To gain more insight into these effects, we have characterized two improved methods for the encapsulation of heterologous molecules inside bacteriophage MS2 viral capsids. First, attaching DNA oligomers to a molecule of interest and incubating it with MS2 coat protein dimers yielded reassembled capsids that packaged the tagged molecules. The addition of a protein stabilizing osmolyte, trimethylamine-N-oxide (TMAO), significantly increased the yields of reassembly. Second, we found that expressed proteins with genetically encoded negatively charged peptide tags could also induce capsid reassembly, resulting in high yields of reassembled capsids containing the protein. This second method was used to encapsulate alkaline phosphatase tagged with a 16 amino acid peptide. The purified encapsulated enzyme was found to have the same Km value and a slightly lower kcat value than the free enzyme, indicating that this method of encapsulation had a minimal effect on enzyme kinetics. This method provides a practical and potentially scalable way of studying the complex effects of encapsulating enzymes in protein-based compartments.
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影响因子：
17.1
作者：
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作者：
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