Virus-Binding Proteins Recovered from Bacterial Culture Derived from Activated Sludge by Affinity Chromatography Assay Using a Viral Capsid Peptide

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.

Construction of a Cloning System for the Mass Production of a Virus-Binding Protein Specific for Poliovirus Type 1

“…The DNA probe for isolation of VBP genomes was constructed based on the N-terminal sequences of VBP (29). The sequence of the constructed DNA probe is 5Ј-GAY ATHCARAARAAYGAYTAYAARTGGTTYCARTTYAAY-3Ј.…”

“…Bacterial culture was developed from activated sludge as described previously (29). One hundred microliters of the bacterial culture derived from activated sludge was suspended in 2 ml of 50 mM EDTA in 50 mM Tris-HCl (pH 8.0).…”

“…The virus-binding ability of the VBP candidate was assayed with enzyme-linked immunosorbent assay (ELISA). Prior to the assay, the solvent of the fusion protein was changed as described previously (29). The fusion protein in the pellet was dissolved in 50 mM carbonate buffer and used for the evaluation of virus-binding ability by ELISA using infectious particles of PV1 as described previously (29).…”

“…Several viruses have a higher resistance to disinfectants than indicator microorganisms (28). A new technology for virus removal needs to be developed in order to prevent the spread of pathogenic viruses in the water environment.In our previous study, virus-binding proteins (VBPs) were successfully recovered from a bacterial culture derived from activated sludge by their affinity with the capsid peptide of poliovirus type 1 (PV1) (29). The isolated VBPs demonstrated the ability to strongly adsorb intact particles of PV1.…”

“…In our previous study, virus-binding proteins (VBPs) were successfully recovered from a bacterial culture derived from activated sludge by their affinity with the capsid peptide of poliovirus type 1 (PV1) (29). The isolated VBPs demonstrated the ability to strongly adsorb intact particles of PV1.…”