Recombinant production, biochemical characterization, and in vitro evaluation of an EWS-FLI1-derived peptide for...

Ewing sarcoma is an aggressive malignancy characterized by the EWS-FLI1 fusion oncoprotein, which remains challenging to target because of its intrinsically disordered nature and lack of intrinsic enzymatic activity. EWSPEP, a peptide derived from the EWS-FLI1 fusion junction region, has previously been investigated in chemically synthesized form; however, its recombinant production has not been reported. In this study, recombinant EWSPEP (rEWSPEP) was expressed in Escherichia coli BL21 Star (DE3) using the pET-45b expression system and purified by Ni²⁺ affinity chromatography, yielding approximately 25-30 mg/L of bacterial culture with an estimated purity of ∼97% based on SDS-PAGE densitometry. Anti-His Western blotting confirmed the presence of the recombinant product, while MALDI-TOF mass spectrometry showed a predominant ion at m/z 8393.8, corresponding to a molecular mass of approximately 8.4 kDa. Functional evaluation in A673 Ewing sarcoma cells demonstrated a concentration- and time-dependent reduction in cell viability following rEWSPEP treatment, accompanied by pronounced morphological alterations. Conditioned media from treated cells showed increased NPM1 and cleaved PARP immunoreactivity, while Western blot analysis showed decreased NPM1 immunoreactivity in cell lysates following treatment. These findings demonstrate the feasibility of recombinant production of EWSPEP and cytotoxic activity of the purified recombinant preparation in A673 cells under the tested conditions. The established recombinant production platform provides a basis for further structural, mechanistic, and functional characterization of rEWSPEP.