Biosynthesis optimization of STEAP1 protein in Pichia pastoris X33 MUT+

• Autor(a) principal: Santos, Margarida Maria Antunes dos
• Data de Publicação: 2016
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/6292
• topic: Biossíntese; Cancro da Próstata; Pichia Pastoris.; Steap1; Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química

Prostate cancer (PCa) is the most common type of cancer in men over the age of 50 in Western countries. The current therapies available are, in many cases, limited and ineffective. Therefore, it is necessary to develop alternative therapies, that can fight specifically and effectively cancer cells. The Six Transmembrane Epithelial Antigen of Prostate 1 (STEAP1) is a protein with six transmembrane domains located in the cell-cell junctions of the secretory epithelium of the prostate. Some studies have reported it as overexpressed in PCa but in normal tissues, its expression is minimal. The structure and cell location of STEAP1 suggest that it may function as a channel for ions. Moreover, it was demonstrated that STEAP1 overexpression induces proliferation of cancer cells. The differential expression of STEAP1 in tissues, associated with its localization, suggests its potential use as a target for cancer therapy. In order to delineate novel strategies for targeting STEAP1 protein, it is necessary to obtain high levels of purified protein. Therefore, the aim of this study is to produce STEAP1 protein in Pichia pastoris X33 Mut+. The use of a eukaryotic system will allow the correct folding of the protein since Pichia pastoris is able to perform post-translation modifications. Our results showed that the target protein is being produced by Pichia pastoris strain and it was identified in the pellet fraction centrifuged at 16000g. Western blot analysis revealed the protein in the correct molecular weight (~ 40 kDa) The best conditions for protein production is a 6 hours fermentation process in BMMH medium with 1.25% methanol and 6% DMSO at 30ºC, 250rpm. The solubilization process should be done using SDS in a concentration of 1-3%.