Preparative Purification of Pseudomonas aeruginosa Bacteriophages via the Combination of Gel-Permeation and Anion-Exchag
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arative Purification of Pseudomonas aeruginosa Bacteriophages via the Combination of Gel-Permeation and Anion-Exchage Chromatography N. N. Landysheva, Y. G. Voronkoa, E. E. Kulikovb, N. N. Sykilindac, and K. A. Miroshnikovc, * a
Institute of Medicine, Peoples’ Friendship University (RUDN University), Moscow, 117198 Russia Winogradsky Institute of Microbiology, Federal Research Center Fundamentals of Biotechnology, Russian Academy of Sciences, Moscow, 117312 Russia c Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia *e-mail: [email protected]
b
Received June 15, 2020; revised June 30, 2020; accepted July 2, 2020
Abstract—We developed and described a method for the purification of Pseudomonas aeruginosa bacteriophages that consists of sequential steps of size-exclusion and anion-exchange chromatography. The technique was tested on five bacteriophages of different size and morphology. Similar results were obtained, which allows the proposed approach to be considered universal and highly scalable. Keywords: bacteriophages, Pseudomonas aeruginosa, anion-exchange chromatography, controlled-pore glass DOI: 10.1134/S0003683820060095
INTRODUCTION Bacteriophages (phages), which were discovered at the beginning of the 20th century, found application in many areas. They are used as antimicrobial agents, phage display aids in peptide studies, and effective vaccination tools and in gene delivery and the specific detection and typing of bacteria. The purity of bacteriophages required for each application varies considerably. In this case, phage preparations intended for medical and food use should contain the minimal amount of bacterial toxins [1–5]. The first successful attempts at the chromatographic purification of phages were undertaken in the 1960–70s [6], but precipitation with polyethylene glycol (PEG) and CsCl density gradient ultracentrifugation later become generally accepted methods for several decades. Such methods ensure the production of bacteriophages of the required purity on a laboratory scale, but they are unacceptable for industrial use due to limitations in scaling and yield and the need for the subsequent removal of CsCl [7]. In recent years, there has been a resurgence of interest in the use of chromatographic methods for phage purification. Numerous studies have shown the effectiveness of anion-exchange chromatography (AEC) on methyl methacrylate monolithic resins; however, the high cost and complexity of carrier regeneration remain significant obstacles to the mass implementation of the method [8–11]. Thus, affordable, scalable,
and efficient methods for the purification of bacteriophages are still needed. The goal of this work is to study the possibility of combined chromatographic purification (gel filtration and AEC) for the preparative purification of bacteriophages. EXPERIMENTAL Bacterial strains and bacteriophages. The characterized bacteriophages were used in the work as model Pseudomonas aeruginosa phages belonging to different groups of the o
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