

@article {8733,
	title = {{Virus-Binding Proteins Recovered from Bacterial Culture Derived from Activated Sludge by Affinity Chromatography Assay Using a Viral Capsid Peptide}},
	journal = {Applied and Environmental Microbiology},
	volume = {70},
	number = {6},
	year = {2004},
	pages = {3434{\textendash}3442},
	abstract = {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 (H 2 N-DNPASTTNKDKL-COOH) was immobilized as a ligand. VBPs exhibited the ability to adsorb infectious particles of PV1 Sabin 1 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. Pollution of water with pathogens has been documented worldwide (13, 16, 17, 25, 37, 41). In particular, chlorine-resis-tant pathogens, such as pathogenic viruses (18), Cryptospo-ridium parvum, and so forth, have raised concerns about out-breaks of waterborne infectious diseases in our society. Actually, pathogenic viruses have been important agents in outbreaks of waterborne diseases (3, 19, 20). Although disin-fection processes using ozone and UV light in conventional water and wastewater treatment systems have contributed to inactivating pathogens, these processes do not always effec-tively inactivate pathogenic viruses because the inactivation efficiency depends on the virus species and turbidity level (8, 11, 36). In addition, membrane technologies, such as microfil-tration and ultrafiltration, are available to remove pathogens (14, 24), but the operation of the membrane separation process has some disadvantages, such as large energy consumption and the nuisance of maintenance. On the other hand, the activated sludge process in wastewa-ter treatment has been well known for removing viruses (9, 23), though the removal efficiency depends on the species and type of viruses (10). Many pathogenic viruses, especially enterovi-ruses, have been observed to be easily captured by sewage sludge (10) and difficult to be eluted (29). Microorganisms in activated sludge yield various polymeric substances including polysaccharides, lipopolysaccharides, proteins, and nucleic acids (38), and these polymers play an important role in the virus adsorption. However, biopolymers in activated sludge contributing to the virus adsorption have not been elucidated. The objective of this study was to discover virus-binding biopolymers from the bacterial culture derived from activated sludge with affinity chromatography. Proteins among biopoly-mers in activated sludge were arbitrarily regarded as the most promising material in this study, because they have a high diversity of configuration. At first, activated sludge bacteria were cultivated in nonselective media, and crude proteins were extracted from bacterial cells harvested. Virus-binding proteins (VBPs) were isolated from the crude proteins by means of their affinity with a viral capsid peptide. The virus-binding ability of VBPs was confirmed by enzyme-linked immunosor-bent assay (ELISA) and a cultural method using HeLa cells. Then, the isolated VBPs were characterized in terms of mo-lecular weight and net surface charges. Amino acid sequences of N termini of VBPs were analyzed, and homology searches for the amino acid sequences of VBPs were conducted.},
	doi = {10.1128/AEM.70.6.3434{\textendash}3442.2004},
	author = {Sano, Daisuke and Matsuo, Takahiro and Omura, Tatsuo}
}
