Two-Step Purification of Elastin-like Polypeptide-fusion Superoxide Dismutase via Hydrophobicity and Thermoresponsiveness

Weiwei Wang^1,2Jinping Chen^1,3Hao Zhu^1,3Huang Aixia¹Dongren Zhou¹Yuchen Wang¹Yang Zhou³Feng Lin^1*Xiangai Dong⁴Yu Wu⁴

• ¹Zhejiang Institute of Freshwater Fisheries, Huzhou, China
• ²College of Biology and Food Engineering, Suzhou University, Suzhou, China
• ³School of Life Sciences, Jiangsu University, Zhenjiang, China
• ⁴The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China

Superoxide dismutase (SOD) catalyzes the dismutation of superoxide radicals to oxygen and hydrogen peroxide, serving as a key antioxidant enzyme with important therapeutic and industrial applications. However, the purification of recombinant SOD remains challenging due to low expression levels and the complexity of traditional purification methods, which involve time-consuming and multi-step chromatography. To address these limitations, we developed a two-step strategy for purifying elastin-like polypeptide (ELP)-tagged human SOD (hSODLEH) leveraging ELP's hydrophobic and thermoresponsive properties. First, foam separation utilized ELP's hydrophobicity to selectively adsorb hSODLEH at the gas-liquid interface, achieving an enrichment ratio of 1.93, protein recovery of 85.67%, enzyme activity enrichment of 2.15, and activity recovery of 93.32% under optimized conditions (0.4 mg/mL protein, 30 °C). Subsequently, inverse transition cycling (ITC) was utilized to further purify hSODLEH by exploiting ELP's thermoresponsiveness, yielding a recovery rate of 91.98% and purification fold of 17.45. The cumulative two-step process resulted in a total yield of 85.84% and overall purification fold of 37.52, yielding the purified hSODLEH with a final purity of approximately 85%. These results demonstrate that ELP-mediated purification offers a scalable and economical alternative to conventional methods. The combination of foam separation and thermal precipitation minimizes the need for expensive chromatography, making this strategy particularly promising for industrial-scale biotechnological applications.