Abstract
MS2 phage-like particles (MS2 PLP) are artificially constructed pseudo-viral particles derived from bacteriophage MS2. They are able to carry a specific single stranded RNA (ssRNA) sequence of choice inside their capsid, thus protecting it against the effects of ubiquitous nucleases. Such particles are able to mimic ssRNA viruses and, thus, may serve as the process control for molecular detection and quantification of such agents in several kinds of matrices, vaccines and vaccine candidates, drug delivery systems, and systems for the display of immunologically active peptides or nanomachines. Currently, there are several different in vivo plasmid-driven packaging systems for production of MS2 PLP. In order to combine all the advantages of the available systems and to upgrade and simplify the production and purification of MS2 PLP, a one-plasmid double-expression His-tag system was designed. The described system utilizes a unique fusion insertional mutation enabling purification of particles using His-tag affinity. Using this new production system, highly pure MS2 PLP can be quickly produced and purified by a fast performance liquid chromatography (FPLC) approach. The system can be easily adapted to produce other MS2 PLP with different properties.
Highlights
MS2 phage-like particles (MS2 PLP) are artificially constructed pseudo-viral particles derived from bacteriophage MS2
MS2 phage-like particles (MS2 PLP) are artificial pseudo-viral particles whose production is based on knowledge of the familiar bacteriophage MS2 (+single stranded RNA (ssRNA), Leviviridae)[1] and the ability of its coat protein dimers to pack specific single-stranded RNA molecules[2,3,4]
Due to these properties and their morphological and physicochemical characteristics, MS2 PLP carrying specific ssRNA molecules can serve as suitable process control virus (PCV) for the detection and quantification of ssRNA viruses within a wide range of samples; for example, human norovirus (NoV), human enterovirus (EV), hepatitis A virus (HAV), hepatitis E virus (HEV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1)[2,5,6,7,8]
Summary
MS2 phage-like particles (MS2 PLP) are artificially constructed pseudo-viral particles derived from bacteriophage MS2. The packaging is driven by dimerization of coat proteins followed by a specific interaction of dimers with a stem-loop structure (called the “pac” site) in ssRNA resulting in spontaneous formation of MS2 PLP These particles are small in size (about 27 nm), non-enveloped, non-infectious, non-replicative, stable pseudo-viral particles with the ability to protect encapsidated ssRNA molecules against the effects of ubiquitous nucleases. The novelty of the present study lies in combination of all the experience acquired so far with the different methods of MS2 PLP construction for developing a new plasmid-driven construction system, which integrates all the advantages and benefits of the previously described systems This is the first report about the construction of single-chain version of the coat protein dimer containing the His-tag capable to successfully form intact His-tagged MS2 PLP that can be purified in less than one hour by FPLC. Presented approach solves the previous problem of introduction of His-tag to structure of the coat protein in parallel with formation of whole intact MS2 PLP
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