An economical and affinity chromatography-free strategy for ricin purification with preserved biological activity.

Ricin, a type II ribosome-inactivating protein derived from Ricinus communis, has attracted considerable attention not only as a potent toxin and biothreat agent but also as a promising component in targeted cancer therapy and immunotoxin development. Its ability to selectively inhibit protein synthesis has been widely exploited in designing antibody-toxin conjugates for therapeutic applications. These diverse applications necessitate the availability of highly purified, biologically active ricin in a cost-effective and scalable manner. In the present study, we report a simplified, economical, and extensible method for ricin purification that eliminates the reliance on affinity chromatography, a major limitation of conventional approaches. Following seed defatting and crude protein extraction, ricin was enriched using sequential ammonium sulfate precipitation at 30% and 50% saturation. SDS-PAGE analysis and in vivo toxicity studies revealed that the 50% ammonium sulfate fraction yielded ricin with minimal contamination from Ricinus communis agglutinin (RCA). This enriched fraction was subsequently subjected to gel filtration chromatography for further purification. The identity and purity of ricin were confirmed by SDS-PAGE, immunoblotting, and LC-MS/MS analyses. Importantly, functional validation in BALB/c mice demonstrated that the LD50 of ricin purified using this optimized method was comparable to that obtained through conventional protocols, indicating preservation of its structural integrity and biological activity. Overall, this study presents a rapid, cost-effective, and scalable purification strategy for producing functionally active ricin, facilitating its broader accessibility for applications in targeted therapeutics, including immunotoxin-based cancer therapy, as well as in toxicology, detection assays, and biodefense research.