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Enhanced stability of detergent-free human native STEAP1 protein from neoplastic prostate cancer cells upon an innovative isolation procedure
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Orientador(es)
Resumo(s)
Background: The STEAP1 is a cell-surface antigen over-expressed in prostate cancer, which contributes to tumor progression and aggressiveness. However, the molecular mechanisms underlying STEAP1 and its structural determinants remain elusive. Methods: The fraction capacity of Butyl- and Octyl-Sepharose matrices on LNCaP lysates was evaluated by manipulating the ionic strength of binding and elution phases, followed by a Co-Immunoprecipitation (Co-IP) polishing. Several potential stabilizing additives were assessed, and the melting temperature (Tm) values ranked the best/worst compounds. The secondary structure of STEAP1 was identified by circular dichroism. Results: The STEAP1 was not fully captured with 1.375 M (Butyl), in contrast with interfering heterologous proteins, which were strongly retained and mostly eluted with water. This single step demonstrated higher selectivity of Butyl-Sepharose for host impurities removal from injected crude samples. Co-IP allowed recovering a purified fraction of STEAP1 and contributed to unveil potential physiologically interacting counterparts with the target. A Tm of ~55 °C was determined, confirming STEAP1 stability in the purification buffer. A predominant α-helical structure was identified, ensuring the protein’s structural stability. Conclusions: A method for successfully isolating human STEAP1 from LNCaP cells was provided, avoiding the use of detergents to achieve stability, even outside a membrane-mimicking environment.
Descrição
POCI‐01‐0145‐FEDER‐ 007491
LA/P/0140/2020
Palavras-chave
Circular dichroism Co-immunoprecipitation Prostate cancer Protein purification STEAP1 Thermal stability Catalysis Molecular Biology Spectroscopy Computer Science Applications Physical and Theoretical Chemistry Organic Chemistry Inorganic Chemistry SDG 3 - Good Health and Well-being