ORIGINAL RESEARCH article
Front. Microbiol.
Sec. Phage Biology
This article is part of the Research TopicAdvancements in Symbiotic Phage Interactions with Microbial Hosts: Bacteria, Algae, and FungiView all 4 articles
Systematic Scale-Up and Enhanced Purification of Marine Cyanophage P-SSP7
Provisionally accepted
- 1Division of Biological Science, Pacific Northwest National Laboratory (DOE), Richland, United States
- 2William R Wiley Environmental Molecular Sciences Laboratory, Richland, United States
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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.
Keywords: Bacteriophage purification workflow, Cryo-EM data collection efficiency, Cyanophage P-SSP7 production scale-up, High-purity phage samples, High-resolution cryo-EM samples, Infectious viral preparation, Local seawater adaptation for scaling, Phage infectivity stability
Received: 26 Dec 2025; Accepted: 31 Jan 2026.
Copyright: © 2026 Bohutskyi, Parvate, Sadler, Chrisler, Cheung and Evans. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Pavlo Bohutskyi
James Evans
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