Virus-binding proteins recovered from bacterial culture derived from activated sludge by affinity chromatography assay using a viral capsid peptide
Virus-binding proteins recovered from bacterial culture derived from activated sludge by affinity chromatography assay using a viral capsid peptide
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DOI：
10.1128/aem.70.6.3434-3442.2004
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发表时间：
2004-06-01
影响因子：
4.4
通讯作者：
Omura, T
中科院分区：
文献类型：
--
作者：
Sano, D;Matsuo, T;Omura, T
病原性病毒对水环境的污染已经引起了人们对病毒性传染病在我们社会中爆发的关注。由于传统的水和废水处理系统不足以有效地抑制或去除致病病毒，因此需要开发一种新的病毒去除技术。在这项研究中，病毒结合蛋白（VBPs）的细菌培养物来自活性污泥中成功地回收。通过将从细菌培养物中提取的粗蛋白质应用于亲和柱来实现VBP的回收，其中来自脊髓灰质炎病毒1型（PV 1）马奥尼株的衣壳蛋白的定制肽（H2 N-DNPASTTNKDKL-COOH）被固定作为配体。VBPs表现出的能力，吸附感染性粒子的PV 1 Sabin I的酶联免疫吸附试验测定。用离子交换色谱法对VBP的表面电荷进行了评价，发现在亲和色谱条件下，大多数VBP分子具有净负电荷。另一方面，计算的等电点暗示亲和柱中的病毒肽也带负电荷。结果，VBP吸附到亲和柱中的病毒肽上时，产生了能够克服静电排斥力的强吸引力。双向电泳结果表明，分离的VBPs含有多种蛋白质，分子量分布较广，但均小于100 kDa。测定了5个VBPs的N端氨基酸序列。在NCBI数据库中对所有蛋白质序列进行N端同源性搜索，结果表明本研究中分离的VBPs是新发现的蛋白质。这些VBP来源于活性污泥中的细菌，可能是稳定的，因为它们存在于废水处理的环境中。因此，可以开发利用VBP作为病毒吸附剂的病毒去除技术，因为可以通过蛋白质克隆技术复制VBP。
The contamination of water environments by pathogenic viruses has raised concerns about outbreaks of viral infectious diseases in our society. Because conventional water and wastewater treatment systems are not effective enough to inactivate or remove pathogenic viruses, a new technology for virus removal needs to be developed. In this study, the virus-binding proteins (VBPs) in a bacterial culture derived from activated sludge were successfully recovered. The recovery of VBPs was achieved by applying extracted crude proteins from a bacterial culture to an affinity column in which a custom-made peptide of capsid protein from the poliovirus type 1 (PV1) Mahoney strain (H2N-DNPASTTNKDKL-COOH) was immobilized as a ligand. VBPs exhibited the ability to adsorb infectious particles of PV1 Sabin I as determined by enzyme-linked immunosorbent assay. The evaluation of surface charges of VBPs with ion-exchange chromatography found that a majority of VBP molecules had a net negative charge under the conditions of affinity chromatography. On the other hand, a calculated isoelectric point implied that the viral peptide in the affinity column was also charged negatively. As a result, the adsorption of the VBPs to the viral peptide in the affinity column occurred with a strong attractive force that was able to overcome the electrostatic repulsive force. Two-dimensional electrophoresis revealed that the isolated VBPs include a number of proteins, and their molecular masses were widely distributed but smaller than 100 kDa. Amino acid sequences of N termini of five VBPs were determined. Homology searches for the N termini against all protein sequences in the National Center for Biotechnology Information (NCBI) database showed that the isolated VBPs in this study were newly discovered proteins. These VBPs that originated with bacteria in activated sludge might be stable, because they are existing in the environment of wastewater treatments. Therefore, a virus removal technology utilizing VBPs as viral adsorbents can be developed, since it is possible to replicate VBPs by protein cloning techniques.