Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, UPM 43400 Serdang, Selangor, Malaysia.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Jul 1;878(21):1855-9. doi: 10.1016/j.jchromb.2010.05.028. Epub 2010 May 24.
M13 is a non-lytic filamentous bacteriophage (phage). It has been used widely in phage display technology for displaying foreign peptides, and also for studying macromolecule structures and interactions. Traditionally, this phage has been purified by cesium chloride (CsCl) density gradient ultracentrifugation which is highly laborious and time consuming. In the present study, a simple, rapid and efficient method for the purification of M13 based on anion exchange chromatography was established. A pre-packed SepFast Super Q column connected to a fast protein liquid chromatography (FPLC) system was employed to capture released phages in clarified Escherichia coli fermented broth. An average yield of 74% was obtained from a packed bed mode elution using citrate buffer (pH 4), containing 1.5 M NaCl at 1 ml/min flow rate. The purification process was shortened substantially to less than 2 h from 18 h in the conventional ultracentrifugation method. SDS-PAGE revealed that the purity of particles was comparable to that of CsCl gradient density ultracentrifugation method. Plaque forming assay showed that the purified phages were still infectious.
M13 是一种非裂解丝状噬菌体（噬菌体）。它已广泛应用于噬菌体展示技术，用于展示外源肽，也用于研究大分子结构和相互作用。传统上，这种噬菌体通过氯化铯（CsCl）密度梯度超速离心法进行纯化，这种方法非常费力且耗时。本研究建立了一种基于阴离子交换层析的简单、快速、高效的 M13 纯化方法。使用连接到快速蛋白质液相色谱（FPLC）系统的预填充 SepFast Super Q 柱从澄清的大肠杆菌发酵液中捕获释放的噬菌体。在 1ml/min 的流速下，使用 pH 值为 4 的柠檬酸钠缓冲液（含 1.5 M NaCl）从填充床模式洗脱中获得了 74%的平均产率。与传统的超速离心法相比，纯化过程大大缩短至不到 2 小时。SDS-PAGE 显示颗粒的纯度与 CsCl 梯度密度超速离心法相当。噬菌斑形成试验表明，纯化的噬菌体仍然具有感染性。
