Browsing by Author "Pedro, Augusto Quaresma Henriques"
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- Biosynthesis, isolation and kinetic characterization of recombinant human catechol-O-methyltransferase from Pichia pastoris strainsPublication . Pedro, Augusto Quaresma Henriques; Passarinha, Luís António Paulino; Queiroz, João António de Sampaio RodriguesCatechol-O-methyltransferase (COMT; EC 2.1.1.6) is a magnesium-dependent enzyme that catalyzes the methylation reaction of different catecholic substrates such as catecholamines, xenobiotic catechols and catecholestrogens. Following the initial characterizations of these enzymes, it was described that they are potentially involved in diverse human disorders. Specifically, as its inhibition has proven to be of great interest in neurologic disorders such as Parkinson's disease, developing inhibitor molecules with increased potency and selectivity may improve the outcome of these patients. These molecules are usually accomplished using structure-based drug design studies that rely on the attainment of highly purified protein quantities. Indeed, challenges in the determination of protein structures are mainly associated with their low natural abundance coupled with the difficulty of obtaining crystals amenable to X-Ray diffraction. In particular, as membrane proteins are naturally embedded in the lipid bilayer, the determination of their structure faces additional difficulties. As it is unrealistic to purify all of these targets from their natural sources, structural biology of proteins usually focus on the recombinant heterologous expression of these proteins onto an expression host. In addition, to isolate the target proteins from the other major host contaminants, equally appropriated purification strategies need to be designed and implemented, mostly using chromatographic procedures. Throughout this entire process, is also important that the developed strategy is able to keep the proteins in a stable and functional active form, thus avoiding its misfolding during biosynthesis and aggregation after its recovery and isolation in the downstream processing. Therefore, the main scope of this work is the development of a straightforward approach that allows the biosynthesis, isolation and purification of recombinant human COMT isoforms in a biologically active form for further application in structural studies or to evaluate their role as potential therapeutic proteins. Specifically, although no single host can provide all the desired properties for recombinant protein biosynthesis, Pichia pastoris is able to perform many post-translational modifications and is cultivated at high cell-densities in moderately cheap media. Therefore, in this work, it was selected for expression of COMT enzymes. On the other hand, the high selectivity often provided by affinity chromatography prompted us to employ it as the main isolation and purification step. The determination of COMT enzymatic activity is greatly important in COMT recombinant research, either to assess COMT activity from recombinant lysates or purified fractions, for detergent-solubilized or unsolubilized samples and for both isoforms. Therefore, a faster and more sensitive analytical method based on HPLC coupled with coulometric detection was developed for quantifying metanephrine in these assays. Then, an integrated strategy for recombinant soluble catechol-O-methyltransferase (SCOMT) biosynthesis onto P. pastoris and purification using immobilized-metal affinity chromatography was implemented where highly purified fractions of this target enzyme were obtained. On the other hand, as heterologous membrane protein overexpression is usually more challenging than soluble proteins and less reports are available in the literature with recombinant human membrane-bound catechol-O-methyltransferase (MBCOMT) than COMT soluble isoform, our work were mostly focused on MBCOMT. Here, we established protocols for MBCOMT expression in Pichia pastoris methanol-induced cultures in baffled shake-flasks and mini-bioreactors. In particular, the optimization of the induction phase using artificial neural networks in mini-biorreactors allowed achieving high levels of biologically active MBCOMT. Then, arginine-affinity chromatography was successfuly applied for the direct capture of MBCOMT from Pichia pastoris lysates and it was recovered in a moderate purified form. Finally, the ongoing work is related to the purification of a hexa-histidine tagged form of MBCOMT using immobilized-metal affinity chromatography. Indeed, despite significant achievements were made concerning the construction of a tagged form of MBCOMT solubilized with an appropriated detergent in a biologically active form, additional stepwise gradients are required to effectively separate MBCOMT from the other contaminants. In conclusion, the progress achieved with this work meets the highly demanding requirements of biophysical techniques, mainly regarding the upstream stage as well as COMT stabilization where moderate to high quantities of catalitically active enzymes were obtained. In particular, coupling the strategy here reported for SCOMT with a final polishing step will probably allow performing structural or bio-interaction studies with this enzyme. Nonetheless, the strategies here described successfully for partial MBCOMT purification need to be improved, especially for immobilized-metal affinity chromatography once it is considered to be highly selective and, thus, it is feasible that after succesful optimization procedures, fractions with high purity will be obtained. Therefore, the strategies here reported with the intensification and optimization of some procedures would possible permit performing structural and bio-interaction studies using the apo-enzymes or complexed with different ligands (cofactors or inhibitors) by Nuclear Magnetic Ressonance, Isothermal Titration Calorimetry or even using Crystallographic experiments.
- Biosynthesis, solubilization and purification of human membrane bound catechol-O-methyltransferase in brevibacillus choshinensis cellsPublication . Pedro, Augusto Quaresma Henriques; Passarinha, Luís António Paulino; Baptista, Cláudio Jorge MaiaAs proteínas membranares constituem cerca de 20 a 30 % de todas as proteínas codificadas pelo genoma de vários organismos. Elevadas quantidades de proteínas num estado de elevada pureza são necessárias quer para estudos farmacológicos, quer para estudos cristalográficos, daí a imperativa necessidade de desenvolver novos sistemas para a sobre-expressão heteróloga de proteínas membranares. Especificamente, nós testámos a aplicação de Brevibacillus choshinensis para a biosíntese da isoforma membranar da catecol-Ometiltransferase humana. No que diz respeito ao processo de produção, obteve-se uma moderada a elevada expressão num meio complexo com um valor de 45 nmol/h/mg para a actividade biológica da hMBCOMT, atingida às 20 horas de cultura a 37 ºC e 250 rpm. No que diz respeito à solubilização da proteína alvo, a eficiência de reconstituição para a hMBCOMT é nula na presença de detergentes iónicos tais como o SDS. No entanto, a aplicação de baixas concentrações de detergentes não-iónicos parece ser ideal para solubilizar a fracção membranar visto que a hMBCOMT recombinante retém elevados valores para a actividade biológica. Dos detergentes testados, a digitonina a 0.5 % (m/v) parece ser o mais adequado. De facto, o método descrito nesta tese é simples e poder-se-á tornar muito útil se aplicado num diagrama global para o isolamento da MBCOMT tendo em vista a sua caracterização bioquímica ou biofísica, onde se destaca a determinação da sua estrutura por cristalografia de raios-X ou estudos de interacção da hMCOMT com inibidores.