Purification of antifreeze proteins from tussah silkworm (Antheraea pernyi) via polyethylene glycol precipitation coupled with aqueous two-phase extraction.

Antifreeze proteins (AFPs) represent specialized proteins capable of inhibiting ice crystal growth and modifying ice crystal morphology. They hold significant potential in biopharmaceuticals, food storage, and cryopreservation. As a characteristic biological resource in China, the tussah silkworm (Antheraea pernyi) is both readily available and rich in AFPs. However, there is currently no efficient, mild, and scalable method for separating and purifying these AFPs. To that end, we adopted a polyethylene glycol (PEG)-based purification strategy coupled with aqueous two-phase extraction (ATPE) to efficiently isolate AFPs from tussah silkworms. First, PEG was used to precipitate the crude extract, thus removing contaminating proteins. The supernatant was then subjected to phase separation by adding saturated ammonium sulfate solution, during which the AFPs were primarily enriched in the upper phase. Through single-factor experiments, we determined the optimal conditions: a PEG molecular weight of 20,000, a PEG concentration of 8 % (w/w), and an ammonium sulfate concentration of 26 % (w/w). Under these conditions, an extraction efficiency of 79.75 ± 5.29 % (n = 3) was achieved. At an AFP concentration of 68.65 mg/mL, the thermal hysteresis activity was 1.82 ± 0.03 °C (n = 3). To confirm the ice-shaping activity, we analyzed the purified product using a nanoliter osmometer, which exhibited hexagonal ice crystals. The target protein was obtained with a purity of 72.56 ± 2.20 % (n = 3). SDS-PAGE analysis showed a single band at 75 kDa, confirming that the protein was substantially enriched. Cytotoxicity assays demonstrated that the extracted AFP exhibited excellent biocompatibility. The coupling of PEG precipitation and ATPE proved effective in rapidly enriching the target protein and removing impurities. This approach provides an application of a potentially scalable strategy for large-scale AFP preparation from tussah silkworm.