Efficient Purification of Cowpea Chlorotic Mottle Virus by a Novel Peptide Aptamer

Viruses. 2023 Mar 7;15(3):697. doi: 10.3390/v15030697.

Abstract

The cowpea chlorotic mottle virus (CCMV) is a plant virus explored as a nanotechnological platform. The robust self-assembly mechanism of its capsid protein allows for drug encapsulation and targeted delivery. Additionally, the capsid nanoparticle can be used as a programmable platform to display different molecular moieties. In view of future applications, efficient production and purification of plant viruses are key steps. In established protocols, the need for ultracentrifugation is a significant limitation due to cost, difficult scalability, and safety issues. In addition, the purity of the final virus isolate often remains unclear. Here, an advanced protocol for the purification of the CCMV from infected plant tissue was developed, focusing on efficiency, economy, and final purity. The protocol involves precipitation with PEG 8000, followed by affinity extraction using a novel peptide aptamer. The efficiency of the protocol was validated using size exclusion chromatography, MALDI-TOF mass spectrometry, reversed-phase HPLC, and sandwich immunoassay. Furthermore, it was demonstrated that the final eluate of the affinity column is of exceptional purity (98.4%) determined by HPLC and detection at 220 nm. The scale-up of our proposed method seems to be straightforward, which opens the way to the large-scale production of such nanomaterials. This highly improved protocol may facilitate the use and implementation of plant viruses as nanotechnological platforms for in vitro and in vivo applications.

Keywords: CCMV-binding peptide; affinity chromatography; affinity extraction; cowpea chlorotic mottle virus; molecular dynamics; nanoparticles; nanotechnology; plant virus; purification; safety issues; ultracentrifugation-free protocol; virus production; virus-like particles.

MeSH terms

  • Aptamers, Peptide* / analysis
  • Aptamers, Peptide* / metabolism
  • Bromovirus*
  • Capsid / metabolism
  • Capsid Proteins / metabolism
  • Nanoparticles*

Substances

  • Aptamers, Peptide
  • Capsid Proteins

Grants and funding

This research received no external funding.