Biosynthesis and isolation of STEAP1 protein from Escherichia coli cells lysates

• Main Author: Pais, João Pedro Vicente
• Publication Date: 2016
• Format: Master thesis
• Language: eng
• Source: Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
• Download full: http://hdl.handle.net/10400.6/5806
• topic: Biossíntese; Cancro da Próstata; Escherichia Coli; Imac.; Proteínas Membranares; Steap1

Prostate cancer is one of the most frequent cancers diagnosed in last years, around the world, and the most common in Europe, mainly in northern and western Europe, where affects more than 200 men in 100.000. In Portugal, the Urology National Association refers to this type of cancer as the most common in adult men but also refers that a precocious detection, increase the treatment success rate to 85%. It is the second cause of death by cancer, and is the most frequent in men over 50 years. The therapies used nowadays are inefficient and limited and do not provide the efficient treatment of this disease. So, new approaches must be assessed and new therapies must be developed to increase the rate of cure. In order to achieve this goal, new molecules arise with a potential role in prostate cancer pathogenesis. The six transmembrane epithelial antigen of the prostate 1 (STEAP1) was found overexpressed in cancer tissues, namely, in prostate cancer cases. The location of this proteins in cell membrane and the six transmembrane domains suggests a role in cell-cell communication, mediating the transport of ions and small molecules. Additionally, its overexpression in cancer cell lines, associated with its topology, makes this protein a promising therapeutic target for immunotherapy. Thus, structural and interaction studies are required, and to achieve that, large amount of highly purified proteins are required. Therefore, the main goal of this work is to develop a laboratorial platform for the STEAP1 protein biosynthesis, solubilization and purification. The obtained results showed that the use of SOB medium, with IPTG at 1.25 mM as inducer after 5h of fermentation and using DMSO at 1.5% (v/v) are the ideal conditions for the biosynthesis of recombinant STEAP1 protein, in small scale. Moreover, applying lactose as inducer may influence the compartmentalization of the target membrane protein and promote its solubility. Concerning the IMAC purification step, a nickel and a cobalt charged columns were used, with different imidazole concentrations in binding step, in order to promote the compete retention of the target biomolecule. Although, several host proteins from E.coli elute along the STEAP1 protein, being necessary additional optimizations in order to obtain a purified STEAP1 sample.