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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.
- New insights for therapeutic recombinant human miRNAs heterologous production: Rhodovolum sulfidophilum vs Escherichia coliPublication . Pereira, Patrícia; Pedro, Augusto; Queiroz, João; Figueiras, Ana R.; Sousa, FaniRNA interference-based technologies have emerged as an attractive and effective therapeutic option with potential application in diverse human diseases. These tools rely on the development of efficient strategies to obtain homogeneous non-coding RNA samples with adequate integrity and purity, thus avoiding non-targeted gene-silencing and related side-effects that impair their application onto pre-clinical practice. These RNAs have been preferentially obtained by in vitro transcription using DNA templates or via chemical synthesis. As an alternative to overcome the limitations presented by these methods, in vivo recombinant production of RNA biomolecules has become the focus in RNA synthesis research. Therefore, using pre-miR-29b as a model, here it is evaluated the time-course profile of Escherichia coli and Rhodovolum sulfidophilum microfactories to produce this microRNA. As the presence of major host contaminants arising from the biosynthesis process may have important implications in the subsequent downstream processing, it is also evaluated the production of genomic DNA and host total proteins. Considering the rapidly growing interest on these innovative biopharmaceuticals, novel, more cost-effective, simple and easily scaled-up technologies are highly desirable. As microRNA recombinant expression fulfills those requirements, it may take the leading edge in the methodologies currently available to obtain microRNAs for clinical or structural studies.
- Trends in protein-based biosensor assemblies for drug screening and pharmaceutical kinetic studiesPublication . Gonçalves, Ana M; Pedro, Augusto; Santos, F M; Martins, Luís M; Baptista, Cláudio; Queiroz, João; Passarinha, L AThe selection of natural and chemical compounds for potential applications in new pharmaceutical formulations constitutes a time-consuming procedure in drug screening. To overcome this issue, new devices called biosensors, have already demonstrated their versatility and capacity for routine clinical diagnosis. Designed to perform analytical analysis for the detection of a particular analyte, biosensors based on the coupling of proteins to amperometric and optical devices have shown the appropriate selectivity, sensibility and accuracy. During the last years, the exponential demand for pharmacokinetic studies in the early phases of drug development, along with the need of lower molecular weight detection, have led to new biosensor structure materials with innovative immobilization strategies. The result has been the development of smaller, more reproducible biosensors with lower detection limits, and with a drastic reduction in the required sample volumes. Therefore in order to describe the main achievements in biosensor fields, the present review has the main aim of summarizing the essential strategies used to generate these specific devices, that can provide, under physiological conditions, a credible molecule profile and assess specific pharmacokinetic parameters.
- Evaluation of Mut(S) and Mut⁺ Pichia pastoris strains for membrane-bound catechol-O-methyltransferase biosynthesisPublication . Pedro, Augusto; Oppolzer, David; Bonifácio, M J; Maia, C J; Queiroz, João; Passarinha, L ACatechol-O-methyltransferase (COMT, EC 2.1.1.6) is an enzyme that catalyzes the methylation of catechol substrates, and while structural and functional studies of its membrane-bound isoform (MBCOMT) are still hampered by low recombinant production, Pichia pastoris has been described as an attractive host for the production of correctly folded and inserted membrane proteins. Hence, in this work, MBCOMT biosynthesis was developed using P. pastoris X33 and KM71H cells in shake flasks containing a semidefined medium with different methanol concentrations. Moreover, after P. pastoris glass beads lysis, biologically and immunologically active hMBCOMT was found mainly in the solubilized membrane fraction whose kinetic parameters were identical to its correspondent native enzyme. In addition, mixed feeds of methanol and glycerol or sorbitol were also employed, and its levels quantified using liquid chromatography coupled to refractive index detection. Overall, for the first time, two P. pastoris strains with opposite phenotypes were applied for MBCOMT biosynthesis under the control of the strongly methanol-inducible alcohol oxidase (AOX) promoter. Moreover, this eukaryotic system seems to be a promising approach to deliver MBCOMT in high quantities from fermentor cultures with a lower cost-benefit due to the cheaper cultivation media coupled with the higher titers tipically achieved in biorreactors, when compared with previously reported mammallian cell cultures.
- Cholinium-Based Good’s Buffers Ionic Liquids as Remarkable Stabilizers and Recyclable Preservation Media for Recombinant Small RNAsPublication . Pedro, Augusto; Pereira, Patrícia; Quental, Maria J.; Carvalho, André P.; Santos, Sérgio M.; Queiroz, João; Sousa, Fani; Freire, Mara G.RNA is a biopolymer of high relevance in the biopharmaceuticals field and in fundamental and applied research; however, the preservation of the RNA stability is still a remarkable challenge. Herein, we demonstrate the enhanced potential of aqueous solutions of self-buffering cholinium-based Good's buffers ionic liquids (GB-ILs), at 20 and 50 % (w/w), as alternative preservation media of recombinant small RNAs. The thermal stability of RNA is highly enhanced by GB-ILs, with an increase of 14 °C in the biopolymer melting temperature - the highest increase observed up to date with ILs. Most GB-ILs investigated improve the stability of RNA at least up to 30-days, both at 25 °C and at 4 °C, without requiring the typical samples freezing. Molecular dynamics simulations were applied to better understand the molecular-level mechanisms responsible for the observed RNA improved stability. The number of IL cations surrounding the RNA chain is similar, yet with differences found for the IL anions, which are responsible for the overall charge of the biopolymer first solvation sphere. No cytotoxicity of the studied solutions containing RNA and ILs at 20 % (w/w) was observed onto two distinct human cell lines, reinforcing their potential to act as preservation media when foreseeing biopharmaceutical applications. Finally, RNA was successfully recovered from the ILs aqueous solutions, without changes in its structural integrity, and the ILs successfully recycled and reused.
- Advances in time course extracellular production of human pre-miR-29b from Rhodovulum sulfidophilumPublication . Pereira, Patrícia; Pedro, Augusto; Tomás, Joana; Baptista, Cláudio; Queiroz, João; Figueiras, Ana; Sousa, FaniThe present study reports the successful production of human pre-miR-29b both intra- and extracellularly in the marine phototrophic bacterium Rhodovulum sulfidophilum using recombinant RNA technology. In a first stage, the optimal transformation conditions (0.025 μg of plasmid DNA, with a heat-shock of 2 min at 35 °C) were established, in order to transfer the pre-miR-29b encoding plasmid to R. sulfidophilum host. Furthermore, the extracellular recovery of this RNA product from the culture medium was greatly improved, achieving quantities that are compatible with the majority of applications, namely for in vitro or in vivo studies. Using this system, the extracellular human pre-miR-29b concentration was approximately 182 μg/L, after 40 h of bacterial growth, and the total intracellular pre-miR-29b was of about 358 μg/L, at 32 h. At the end of the fermentation, it was verified that almost 87 % of cells were viable, indicating that cell lysis is minimized and that the extracellular medium is not highly contaminated with the host intracellular ribonucleases (RNases) and endotoxins, which is a critical parameter to guarantee the microRNA (miRNA) integrity. These findings demonstrate that pre-miRNAs can be produced by recombinant RNA technology, offering novel clues for the production of natural pre-miRNA agents for functional studies and RNA interference (RNAi)-based therapeutics.
- Integrated Extraction-Preservation Strategies for RNA Using Biobased Ionic LiquidsPublication . Quental, Maria V.; Pedro, Augusto; Pereira, Patrícia; Sharma, Mukesh; Queiroz, João; Coutinho, João A.P.; Sousa, Fani; Freire, Mara G.The ubiquitous instability of RNA along with issues associated with its purity degree have been preventing its widespread use as low-cost biotherapeutics. On the basis of the well-known capacity of amino acids to specifically interact with RNA when used as chromatographic ligands, a set of amino-acid-based ionic liquids (AA-ILs) was herein investigated, both to act as preservation media and as phase-forming agents of aqueous biphasic systems (ABS). This set of strategies was combined with the goal of developing integrated extraction-preservation platforms. AA-ILs comprising the cholinium cation and anions derived from l-lysine ([Ch][Lys]), l-arginine ([Ch][Arg]), l-glutamic acid ([Ch][Glu]), and dl-aspartic acid ([Ch][Asp]) were studied. It is shown that the stability of RNA is preserved in aqueous solutions of the studied AA-ILs, even in the presence of ribonucleases (RNases). Furthermore, almost all the investigated AA-ILs display no cytotoxicity onto two distinct human cell lines. After identifying the most promising ILs, ABS formed by AA-ILs and polypropylene glycol with a molecular weight of 400 g mol–1 (PPG 400) were investigated as extraction and purification platforms for RNA. Both with pure RNA and bacterial lysate samples, RNA is successfully extracted to the IL-rich phase without compromising its integrity and stability. On the basis of these results, the integrated extraction-preservation process for RNA is finally demonstrated. RNA is initially extracted from the bacterial lysate sample using ABS, after which the IL-rich phase can be used as the preservation medium of RNA up to its use. RNA can be then recovered from the IL-rich phase by ethanol precipitation, and the ABS phase-forming components recovered and reused. Although improvements in the purity level of RNA are still required, the approach here reported represents a step forward in the development of sustainable processes to overcome the critical demand of high-quality/high-purity RNA to be used as biotherapeutics.
- Pichia pastoris: a recombinant microfactory for antibodies and human membrane proteinsPublication . Gonçalves, A M; Pedro, Augusto; Maia, C J; Sousa, Fani; Queiroz, João; Passarinha, L ADuring the last few decades, it has become evident that the compatibility of the yeast biochemical environment with the ability to process and translate the RNA transcript, along with its capacity to modify a translated protein, are relevant requirements for selecting this host cell for protein expression in several pharmaceutical and clinical applications. In particular, Pichia pastoris is used as an industrial host for recombinant protein and metabolite production, showing a powerful capacity to meet required biomolecular target production levels in high-throughput assays for functional genomics and drug screening. In addition, there is a great advantage to using P. pastoris for protein secretion, even at high molecular weights, since the recovery and purification steps are simplified owing to relatively low levels of endogenous proteins in the extracellular medium. Clearly, no single microexpression system can provide all of the desired properties for human protein production. Moreover, chemical and physical bioprocess parameters, including culture medium formulation, temperature, pH, agitation, aeration rates, induction, and feeding strategies, can highly influence product yield and quality. In order to benefit from the currently available wide range of biosynthesis strategies using P. pastoris, this mini review focuses on the developments and technological fermentation achievements, providing both a comparative and an overall integration analysis. The main aim is to highlight the relevance and versatility of the P. pastoris biosystem to the design of more cost-effective microfactories to meet the increasing demands for recombinant membrane proteins and clinical antibodies for several therapeutic applications.
- 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.
- Brain-Targeted Delivery of Pre-miR-29b Using Lactoferrin-Stearic Acid-Modified-Chitosan/Polyethyleneimine PolyplexesPublication . Pereira, Patrícia; Barreira, Maria; Cruz, Carla; Tomás, Joana; Luís, Ângelo; Pedro, Augusto; Queiroz, João; Sousa, FaniThe efficacy of brain therapeutics is largely hampered by the presence of the blood-brain barrier (BBB), mainly due to the failure of most (bio) pharmaceuticals to cross it. Accordingly, this study aims to develop nanocarriers for targeted delivery of recombinant precursor microRNA (pre-miR-29b), foreseeing a decrease in the expression of the BACE1 protein, with potential implications in Alzheimer's disease (AD) treatment. Stearic acid (SA) and lactoferrin (Lf) were successfully exploited as brain-targeting ligands to modify cationic polymers (chitosan (CS) or polyethyleneimine (PEI)), and its BBB penetration behavior was evaluated. The intracellular uptake of the dual-targeting drug delivery systems by neuronal cell models, as well as the gene silencing efficiency of recombinant pre-miR-29b, was analyzed in vitro. Labeled pre-miR-29b-CS/PEI-SA-Lf systems showed very strong fluorescence in the cytoplasm and nucleus of RBE4 cells, being verified the delivery of pre-miR-29b to neuronal cells after 1 h transfection. The experiment of transport across the BBB showed that CS-SA-Lf delivered 65% of recombinant pre-miR-29b in a period of 4 h, a significantly higher transport ratio than the 42% found for PEI-SA-Lf in the same time frame. Overall, a novel procedure for the dual targeting of DDS is disclosed, opening new perspectives in nanomedicines delivery, whereby a novel drug delivery system harvests the merits and properties of the different immobilized ligands.