Engineering and Purification of Native-Like Recombinant TBEV/LGTV E and NS1 Antigens Using SUMO Fusion for Future Differential Diagnostic Applications

Tick-borne encephalitis virus (TBEV) remains a significant public health concern across Europe, Russia, and parts of Asia. Although vaccination programs are widely implemented, clinical differentiation between vaccinated individuals and those with natural TBEV infection remains challenging. In this study, we investigate the envelope (E) and non-structural protein 1 (NS1) of TBEV-alongside their Langat virus (LGTV) orthologs-as serological markers capable of discriminating infection-induced from vaccine-induced antibody responses. We cloned and expressed four constructs: TBEV‑E, TBEV‑NS1, LGTV‑E, and LGTV‑NS1, using a SUMO-His tag system in Escherichia coli . Protein expression was validated via SDS‑PAGE and anti‑His Western blotting. High-level soluble expression and Ni²⁺-NTA purification were achieved, followed by dialysis-based refolding. The SUMO tag was precisely removed using ULP‑1 protease, yielding native E (~ 50 kDa) and unprocessed NS1 (~ 60 kDa) or complete degradated, as confirmed by Western blot analysis. Successful SUMO cleavage and native protein elution occurred only under denaturing lysis (guanidine buffer), demonstrating method-specific efficiency. The purified recombinant E and NS1 proteins could be used for solid foundation for ELISA assay development designed to distinguish antibody responses resulting from natural TBEV infection versus vaccination. Our work offers a scalable and optimized protein purification methods for producing pure native TBEV structural proteins and supports the development of diagnostic platforms that enhance epidemiological surveillance and clinical decision-making in TBE-endemic regions.