Design of an one-step platform purification of STEAP1 using Hydrophobic Interaction Chromatography

• Autor(a) principal: Monteiro, Diogo José Pinheiro
• Data de Publicação: 2018
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/9773
• topic: Cancro da Próstata; Cromatografia de Interação Hidrofóbica; Purificação; Steap1; Domínio/Área Científica::Ciências Médicas::Ciências Biomédicas

Prostate cancer is one of the most lethal and prevalent carcinoma among elder men worldwide. Currently, prostate cancer diagnosis based on prostate-specific antigen levels is unspecific and with limited efficient, mainly in advanced stages of cancer. Thus, there is a need to identify and characterize specific and reliable protein biomarkers for prostate cancer. Six transmembrane epithelial antigen of the prostate 1 (STEAP1) is a transmembrane protein whose high expression levels were correlated with PCa. STEAP1 may take part in intracellular and intercellular communication in cancer cells by modulating cell proliferation and tumor invasiveness through its potential activity as ion channel or transporter. So, the characterization of STEAP1 structure might allow the design of specific inhibitors that decrease and modulate its oncogenic function. The structural and functional studies require high purified amounts of protein, which can be obtained through a recombinant production of human STEAP1 protein integrated with a properly chromatographic strategy. In this work, the performance of Octyl- and Butyl-Sepharose were evaluated according to binding and elution conditions required for STEAP1 isolation from cell lysates, obtained in mini-bioreactor Pichia pastoris X33 methanol-induced cultures. The concentration of sodium phosphate buffer and monosodium phosphate plus sodium chloride in the equilibration buffer was optimized in order to promote a complete STEAP1 adsorption on the hydrophobics supports. Succinctly, a higher retention of STEAP1 was observed with concentrations above 500 mM of sodium phosphate buffer and monosodium phosphate plus sodium chloride, pH 8.0. If the adsorption is achieved at high concentrations of sodium phosphate buffer, the elution must be performed with increasing concentrations of Triton X-100 in 50 mM phosphate buffer. The obtained results indicate that the exposition of membrane binding domains of STEAP1 to Octyl- and Butyl- Sepharose requires high salt concentrations due the strong interactions established between them. However, after its complete adsorption, STEAP1 elution requires chaotropic agents such as detergents. Although application of HIC in the purification of integral membrane proteins are uncommon, the obtained results in the development of this dissertation indicate that the use of traditional hydrophobic matrices may open a promising alternative for the isolation of STEAP1 from cell lysates.