Optimizing the synthesis and purification of MS2 virus like particles

Khadijeh Hashemi,Bizhan Malaekeh-Nikouei,Mohammad Reza Bassami,Mohammad Reza Ahmadian,Amir Afkhami-Goli,Mohammad Mahdi Ghahramani Seno,Hesam Dehghani

doi:10.1038/s41598-021-98706-1

Abstract

Introducing bacteriophage MS2 virus-like particles (VLPs) as gene and drug delivery tools increases the demand for optimizing their production and purification procedure. PEG precipitation method is used efficiently to purify VLPs, while the effects of pH and different electrolytes on the stability, size, and homogeneity of purified MS2 VLPs, and the encapsulated RNA sequences remained to be elucidated. In this regard, a vector, capable of producing VLP with an shRNA packed inside was prepared. The resulting VLPs in different buffers/solutions were assessed for their size, polydispersity index, and ability to protect the enclosed shRNA. We report that among Tris, HEPES, and PBS, with or without NaNO3, and also NaNO3 alone in different pH and ionic concentrations, the 100 mM NaNO3-Tris buffer with pH:8 can be used as a new and optimal MS2 VLP production buffer, capable of inhibiting the VLPs aggregation. These VLPs show a size range of 27-30 nm and suitable homogeneity with minimum 12-month stability at 4 °C. Moreover, the resulting MS2 VLPs were highly efficient and stable for at least 48 h in conditions similar to in vivo. These features of MS2 VLPs produced in the newly introduced buffer make them an appropriate candidate for therapeutic agents’ delivery.

Highlights

Introducing bacteriophage MS2 virus-like particles (VLPs) as gene and drug delivery tools increases the demand for optimizing their production and purification procedure
The MS2 VLP is a 27 nm spherical RNA bacteriophage virus-like particle, consisting of 180 identical 129-amino acid coat protein subunits of ~ 14 kDa that form the icosahedral face for a T = 3 surface lattice[5,6,7]
To confirm the expression of the coat protein of VLPs, ~ 14 kDa disassembled protein of purified MS2 VLPs was separated by electrophoresis on 12.5% acrylamide gel and detected using specific antibody and Western blot method

Summary

Introduction

Introducing bacteriophage MS2 virus-like particles (VLPs) as gene and drug delivery tools increases the demand for optimizing their production and purification procedure. PEG precipitation method is used efficiently to purify VLPs, while the effects of pH and different electrolytes on the stability, size, and homogeneity of purified MS2 VLPs, and the encapsulated RNA sequences remained to be elucidated In this regard, a vector, capable of producing VLP with an shRNA packed inside was prepared. The interaction with the 5’-terminus of the so-called pac site has been used as a preferred strategy to encapsulate different c argoes[2,8] In this respect, it is very important to understand the behavior and assembly of virus-like particles and their aggregation and stability in different conditions. In the recombinant production of MS2 VLPs, upon the transformation of plasmids expressing MS2 coat protein and a part of maturase into the bacteria, the most appropriate pH and buffers to support the stability and homogeneity of VLPs are sought after[12,34,35,36]

Methods

Results

Conclusion