Systematic scale-up and enhanced purification of marine cyanophage P-SSP7

TL;DR

Researchers developed a scalable and efficient method to produce high-quality marine cyanophage P-SSP7 for structural and functional studies.

Contribution

An integrated methodology for scaling up and purifying cyanophage P-SSP7 to meet cryo-EM quality standards is introduced.

Findings

The method achieves infectious phage titers exceeding 3 × 10¹² units/mL with >95% purity.

Cryo-EM data collection time is reduced 60-fold through optimized purification.

Phage infectivity is retained at 68% after 3 months and 23% after 6 months at 4°C.

Abstract

Cyanophages represent important models for understanding virus-host interactions, yet high-resolution structural, functional, and dynamical studies remain relatively few due to challenges with preparing enough sample of sufficient quality for cryo-electron microscopy (cryo-EM) and multi-omics studies. Here we developed an integrated methodology for scaling production of the model cyanophage P-SSP7 from laboratory maintenance volumes (5–100 mL) to production scales (up to 40 L) while dramatically improving the quality of phage preparation for structural applications. Our systematic approach integrates host cultivation using adaptation to local seawater to reduce production costs, optimized infection protocols to maximize infectious titer yields, and multi-stage purification workflows specifically designed for cryo-EM quality requirements. The final methodology consistently produces infectious phage titers exceeding 3 × 1012 units/mL with recoverable yields of 1013 total infectious units and >95% purity validated by cryo-EM at each optimization step. Most significantly, this approach achieves a 60-fold reduction in cryo-EM data collection time between the initial and final optimization steps by increasing usable particles per field of view for single particle analysis. Overall, our final preparations demonstrate robust phage stability, retaining 68% infectivity after 3 months and 23% after 6 months at 4 °C. This workflow moves cyanophage culturing and downstream structural studies from specialized, resource-intensive endeavors toward routine research capability and establishes an adaptable framework for scaling production that can be applied to other host-virus systems.

Figures