One-plasmid double-expression His-tag system for rapid production and easy purification of MS2 phage-like particles

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.

Bacteriophage MS2 displays unreported capsid variability assembling T = 4 and mixed capsids

“…Although particles have been made available commercially for diseases like Hepatitis C and Enterovirus (Armoured RNA®), the sequences of the packaged RNA are not publicly available. Various particle purification methodologies have been suggested and single vectors have been described that allow for the purification of MS2 virus‐like particles with user‐specified RNA sequences using affinity chromatography (Mastico et al , ; Mikel et al , ). We therefore created a single plasmid vector with the CP‐His‐CP MS2 dimer (Peabody and Lim, ; Mikel et al , ) which was co‐expressed with maturation protein and a 155 bp heterologous RNA sequence incorporating a modified MS2 stem‐loop (c‐variant pac site ) (Wei et al , ) (Supplementary Figs A–C, and A).…”

“…Cells were harvested by centrifugation at 3220× g at 4°C, followed by resuspension in 4 ml of sonication buffer (50 mM Tris‐HCl pH 8.0, 5 mM MgCl 2 , 5 mM CaCl 2 , 100 mM NaCl) described previously (Mikel et al , ) and supplemented with 700U RNAse A (Qiagen), 2500U BaseMuncher (Expedeon) and 200U Turbo DNAse (Thermo Fisher Scientific). The cells were then lysed by sonication (50% amplitude, 30 seconds on, 30 seconds off, 4 pulses) at 4°C.…”

“…For the wild‐type MS2 construct, the filtered supernatant was applied to a glycerol gradient from 10 to 40% 160,000× g (30 000 rpm) 20 h in a SW40 rotor. For CP‐His‐CP MS2 constructs, all buffers used were from previously described methods (Mikel et al , ). The filtered supernatant was mixed 1:1 with 2X binding buffer (100 mM NaH 2 PO 4 ∙H 2 O pH 8.0, 30 mM imidazole, 600 mM NaCl) and loaded onto a 5 ml HiTrap TALON Crude column (GE Healthcare Life Sciences) with a 1 ml HiTrap Heparin HP column (GE Healthcare Life Sciences) in series via FPLC (ÄKTA Pure, GE Healthcare Life Sciences).…”

A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics

“…bacteriophage VLPs carrying the SARS-CoV-2 N-gene were produced in Escherichia coli from an expression plasmid using protocols described previously and modified to transcribe and package the RNA for the SARS-CoV-2 N protein ( Fig. 1a) 13,14 . The assembled MS2-SARS-CoV-2 VLPs were purified and treated with DNase and RNase, to ensure the preparation was free from template DNA and RNA contamination.…”

A new role for Biofoundries in rapid prototyping, development, and validation of automated clinical diagnostic tests for SARS-CoV-2

“…The N gene was cloned into a previously described plasmid backbone (Addgene #128233) using Type IIs assembly. The sequence verified (Eurofins Genomics) The protein purification workflow is based on previously described work [12], [30]. All protein purification steps were performed at 4 °C.…”