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- Isolation of the Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) using Gellan Gum MicrospheresPublication . Silva, João Pedro Batista da; Passarinha, Luís António Paulino; Sousa, Ângela Maria Almeida de; Ferreira, Jorge Daniel BarrocaProstate cancer is one of the most frequent cancers diagnosed in males, predicted to become the most prevalent malignancy in upcoming years. Current therapies have limited efficacy and are not suitable to mitigate the alarming incidence and mortality rates, especially in later hormone-refractory stages. In this manner, it is crucial the development of novel approaches. Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) is a protein highly overexpressed in PCa predicted to function as a transmembrane transporter and ionic channel, to play a role in intercellular communication between tumor cells and has been associated with the generation and propagation of high levels of reactive oxygen species. Consequently, STEAP1 leads to increased cellular proliferation and tumor aggressiveness. Furthermore, STEAP1 is mostly absent from normal tissues, suggesting a specificity for the cancer microenvironment, indicating that STEAP1 could have potential as a biomarker and therapeutic target. However, to explore this potential it is first necessary to isolate stable, bioactive and pure protein fractions of high concentration. Thus, we prepared calciumand nickel-crosslinked gellan gum microspheres through a water-in-oil emulsion with 1.41% (w/v) gellan at 750 rpm and 100ºC. The microspheres were characterized trough FTIR, SEM and EDX, ensuring adequate size, morphology, and crosslinking. The microspheres were used to capture STEAP1 solubilized in 0.1% (v/v) DM derived from Komagataella pastoris X33 Mut+ mini-bioreactor lysates using two different approaches, exploring ionic and affinity interactions. The ionic strategy presented the best results and consisted of STEAP1 capture in 10 mM MES buffer at pH 6.2. The target was then eluted by electrostatic repulsion with 10 mM Tris supplemented with 500 mM NaCl at pH 11. These optimized conditions allowed the recovery of STEAP1 in a single step, eliminating heterologous proteins in intermediary steps. The clarified fraction was polished by coupling with a Co-immunoprecipitation step. This technique was able to decomplex STEAP1-gellan gum complexes formed during the batch clarification. This innovative integration results in a monomeric STEAP1 fraction with high purity degree.