Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification

Abstract

Bacteriophages may be readily concentrated from crude lysates of infected bacteria after addition of polyethylene glycol (PEG). A general procedure is presented which can be used for lysate volumes of 17 liters or more with nearly quantitative recovery of infectivity over a wide range of phage titers (10 5 to 10 13 PFU/ml). All the bacteriophages tested (λ, T4, T7, P22, fd, φX174, R17) are efficiently removed from solution by simple settling at concentrations of PEG 6000 between 2% and 10%. Bacteriophage pellets are redissolved in a small amount of buffer, allowing 100-fold concentration of the original lysate. While lower molecular weight polyethylene glycols are much less effective for concentrating bacteriophages than the PEG 6000 used, the efficiency is relatively insensitive to changes in pH and ionic strength. Asymmetric particles (tobacco mosaic virus and bacteriophage fd) are especially susceptible to PEG, and they can be purified from more symmetrical particles at low PEG 6000 concentrations (2% or less). Although the exact mechanism by which bacteriophages can be concentrated with PEG is unknown, a phase partition rather than a normal precipitation reaction seems to be involved, since the fraction of infective phages removed from solution by a fixed concentration of PEG is nearly invariant to changes in bacteriophage concentration over as much as a 10 8-fold range. Extension of this method to concentration and purification of other viruses and nucleic acids, as well as some preliminary mechanistic studies, are discussed.