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Published on: March 25, 2010
Purification of filamentous bacteriophage M13 by expanded bed anion exchange chromatography
Tau Chuan Ling1, Chee Kin Loong, Wen Siang Tan
1Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.
Abstract:
In this paper, we investigated the development of a simplified and rapid primary capture step for the recovery of M13 bacteriophage from particulate-containing feedstock. M13 bacteriophage, carrying an insert, was propagated and subsequently purified by the application of both conventional multiple steps and expanded bed anion exchange chromatography. In the conventional method, precipitation was conducted with PEG/NaCl, and centrifugation was also performed. In the single step expanded bed anion exchange adsorption, UpFront FastLine 20 (20 mm i.d.) from UpFront Chromatography was used as the contactor, while 54 ml (Ho = 15 cm) of STREAMLINE DEAE (rho = 1.2 g/cm3) from Amersham Pharmacia Biotechnology was used as the anion exchanger. The performance of the two methods were evaluated, analysed, and compared. It was demonstrated that the purification of the M13 bacteriophage, using expanded bed anion exchange adsorption, yielded the higher recovery percentage, at 82.86%. The conventional multiple step method yielded the lower recovery percentage, 36.07%. The generic application of this integrated technique has also been assessed.
Insights
Simplified M13 bacteriophage purification using expanded bed anion exchange chromatography achieved 82.86% recovery. This rapid primary capture method offers a significant improvement over conventional multi-step purification techniques for M13 bacteriophage recovery.
Area of Science:
- Biotechnology
- Virology
- Chromatography
Background:
- M13 bacteriophage is a valuable tool in molecular biology and biotechnology.
- Efficient purification is crucial for its applications.
- Existing purification methods can be complex and time-consuming.
Purpose of the Study:
- To develop a simplified and rapid primary capture method for M13 bacteriophage recovery.
- To compare the efficiency of expanded bed anion exchange chromatography with conventional purification methods.
- To assess the generic applicability of the developed technique.
Main Methods:
- M13 bacteriophage was propagated and purified using two methods: conventional multi-step purification and single-step expanded bed anion exchange chromatography.
- Conventional method involved PEG/NaCl precipitation and centrifugation.
- Expanded bed anion exchange chromatography utilized UpFront FastLine 20 contactor and STREAMLINE DEAE anion exchanger.
Main Results:
- Expanded bed anion exchange chromatography achieved a significantly higher recovery percentage of 82.86% for M13 bacteriophage.
- The conventional multi-step method yielded a lower recovery percentage of 36.07%.
- The integrated technique demonstrated potential for generic application.
Conclusions:
- Expanded bed anion exchange chromatography provides a more efficient and higher-yielding method for M13 bacteriophage primary capture.
- This simplified approach reduces purification time and complexity.
- The developed method shows promise for broad application in bacteriophage recovery.
