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- Unveiling Biopathways from the Biosynthesis Towards the Purification of STEAP1 Protein New Insights on Stability, Structure, and FunctionPublication . Ferreira, Jorge Daniel Barroca; Passarinha, Luís António Paulino; Baptista, Cláudio Jorge Maia; Silva, Teresa Sacadura SantosIn 2020, more than 1.4 million new prostate cancer (PCa) cases were diagnosed in man worldwide with approximately 375,000 mortal victims. Nowadays, the lack of prognostic and predictive biomarkers associated to the poor effectiveness and specificity of the available therapies, demand the identification of novel and highly specific agents aiming to establish a tailor-made theragnostic strategy targeting PCa. The Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) is an integral six-transmembrane protein over-expressed in PCa, in contrast with non-tumoral tissues and vital organs, wherein the expression levels of STEAP1 are reduced or absent. Based on the amino-acid sequence, secondary structure, transmembrane topology, and cellular localization, STEAP1 play a crucial role in cellular communications and cell adhesion processes as a transporter protein and ion channel, and in the stimulation of cell growth by increasing the intracellular levels of reactive oxygen species, ultimately contributing to tumor progression and aggressiveness. Moreover, it is capable of reducing extracellular metal-ion complexes and molecular oxygen through the biological behavior of heme-binding site, playing a role in metal metabolism. These biological features and promising results of several in vitro and in vivo studies emphasize the usefulness of STEAP1 as a promising biomarker or target for anti-cancer therapies. However, high-resolution structures for STEAP1 are not available, highlighting the lack of detailed structural and functional knowledge on the target protein, which impairs the complete understanding of its biological role in PCa. So far, only one structure of full-length human STEAP1 was solved by cryogenic-Electron Microscopy (cryo-EM) (PDB code: 6y9B, 2.97 Å resolution). Thus, this Thesis proposes the implementation of a biotechnology laboratorial hands-on platform, starting with the biosynthesis towards the purification of the STEAP1 protein, to ultimately discover antagonist biological molecules or inhibitory drugs that will be able to block the oncogenic effects triggered by the target. Firstly, we expressed the full-length human native STEAP1 in Lymph Node Carcinoma of the Prostate (LNCaP) cell cultures followed by its isolation using a hydrophobic Butyl-Sepharose resin and co-immunoprecipitation as polishing strategy to recover a STEAP1 sample with a high degree of purity. The purified sample was highly thermostable in the absence of a membrane-mimicking environment, hence, not requiring the addition of a specific buffer or additive to preserve its α-helical secondary structure during purification as shown by circular dichroism. Then, we developed a mini-bioreactor strategy to scale-up the biosynthesis yield of STEAP1 using recombinant Komagataella pastoris X-33 Mut+ methanol-induced cultures. The influence of glycerol feeding profiles – constant, gradient, and exponential - in the STEAP1 expression levels was evaluated. The supplementation with 6% (v/v) DMSO and 1 M Proline increased the levels of stable N-glycosylated protein. The biological role of STEAP1 was validated in PCa neoplastic LNCaP and PC3 cell lines, showing an increment in cell proliferation. Then, a suitable solubilization and purification strategy for recombinant STEAP1 was explored. The target was effectively extracted and solubilized into 0.1% (v/v) Nonidet P-40 (NP-40) and η-Decyl-β-D-Maltopyranoside (DM) detergents and further isolated using Phenyl-Sepharose and Nickel-loaded resins as capture step. A combination with Q-Sepharose matrix was effective as polishing stage and allowed obtaining the target protein with high degree of purity and fully encapsulated in detergent micelles. Finally, the STEAP1 was recombinantly over-expressed in Human Embryonic Kidney (HEK)-293 cells. The solubilizing effectiveness of a wide-range of detergents and non-micellar agents using high-throughput technology was assessed. Equally, η-Dodecyl-β-D-Maltopyranoside (DDM)+Cholesteryl Hemisuccinate (CHS) and Lauryl Maltose Neopentyl Glycol (LMNG)+CHS provided increased recovery of fully solubilized STEAP1. The target was successfully purified using Talon Co2+ resin coupled to Fluorescence-based Size Exclusion Chromatography (FSEC), in a sample with high protein quality, degree of purity, and with a protein mass of approximately 2 μg. The purified sample was analyzed by EM which showed several acceptable STEAP1 particles within the expected size for multimers. Altogether, an optimized and reproducible bioprocess for the biosynthesis of both native and recombinant forms of STEAP1 protein and their successful purification in samples with increased degree of purity was established. These results are the starting point to structural studies aiming at high-resolution structures of the STEAP1 protein, which remains to be disclosed.
- Isolation and purification of STEAP1 protein fragment in Escherichia coli cells: a potential target for prostate cancerPublication . Ferreira, Jorge Daniel Barroca; Batista, Cláudio Jorge Maia; Passarinha, Luís António PaulinoProstate cancer is the second most prevalent type of cancer worldwide and the sixth cancer-related death. The current applied therapies in advanced cancer stages are limited and not completely efficient. Thus, it is necessary to develop alternatives using, for example, molecular biology vanguard technologies that allow to identify genes that encode transmembrane proteins, abundantly expressed in cancer tissues. The six-transmembrane epithelial antigen of the prostate (STEAP1) was recognized as a protein presenting a structure with six transmembrane domains connected by extracellular loops. It is commonly located in plasma membrane, for example in communication junctions or even in endosomal membranes, suggesting a role as ion channel or transporter protein in intracellular communication between tumor cells. In association with its high specificity and significant expression levels in prostatic cancer tissues, STEAP1 is a promising candidate to be imposed as a therapeutic target, especially for immunotherapy. However, the impossibility of large-scale production hinders the development of structural and biointeraction studies, not only to establish the tridimensional structure of STEAP1 but also to understand its vivo behavior. Therefore, the main objectives consisted in establish the culture medium formulation and optimal fermentation conditions in order to achieve a maximum yield for the STEAP1 first loop (STEAP11-142) biosynthesis, besides access several conditions inherent to immobilized metal affinity chromatography (IMAC) to obtain considerable levels of purified peptide. The main results showed that TB medium fermentation at 37 ºC, 250 rpm and pH 7.2 over 8 hours increased the STEAP11-142 biosynthesis levels. Moreover, Triton X-100 at 1 % (v/v) demonstrated to be the most effective detergent for protein recovery. This result was validated by Western-blotting analysis which revealed a single immunoreactive band with the expected molecular weight (17-25 kDa). Concerning the purification step by IMAC, we tested two resins charged with nickel and cobalt. The results lead to a typical chromatographic profile where the target fragment was completed retained. Nevertheless, a considerable amount of heterologous proteins from Escherichia coli (E. coli) host co-eluted with the STEAP11-142 fragment, being necessary to develop an alternative elution strategy and optimize the purity of the target fractions.