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Published on: October 14, 2011
Characterization and purification of bacteriophages using chromatofocusing
Kurt Brorson1, Hong Shen, Scott Lute
1Center for Drug Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD 20993, USA.
Journal of Chromatography. A
|September 10, 2008
Summary
Chromatofocusing effectively purifies and characterizes bacteriophages (viruses that infect bacteria) using defined buffers and specific column packing. This method provides rapid isoelectric point estimation and high recovery rates for live viruses.
Area of Science:
- Virology
- Biochemistry
- Chromatography
Background:
- Bacteriophages are crucial for virus filter testing.
- Traditional chromatofocusing uses polyampholyte buffers, which can be less defined.
- Minimizing band broadening is key for accurate characterization.
Purpose of the Study:
- To characterize and purify bacteriophages using chromatofocusing.
- To evaluate the efficacy of chemically defined eluent buffers.
- To determine bacteriophage isoelectric points and assess purification efficiency.
Main Methods:
- Application of chromatofocusing with chemically defined eluent buffers.
- Selection of chromatographic column packings for surface adsorption.
- Utilizing plaque-forming assays to determine live phage recovery.
Main Results:
- Bacteriophages (phiX174, PR772, PP7) focused into narrow bands in a pH gradient.
- Live phage recovery ranged from 15% to nearly 100%.
- Approximate isoelectric points were determined for purified and crude bacteriophage preparations.
Conclusions:
- Chromatofocusing with defined buffers is a rapid and simple method for bacteriophage characterization.
- The technique allows for efficient purification of bacteriophages and potentially other viruses.
- This method aids in obtaining approximate isoelectric points and assessing purity.
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