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Evaluation of Mut(S) and Mut⁺ Pichia pastoris strains for membrane-bound catechol-O-methyltransferase biosynthesis

Publication . Pedro, Augusto; Oppolzer, David; Bonifácio, M J; Maia, C J; Queiroz, João; Passarinha, L A

Catechol-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.

2015-04Journal article

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Biosynthesis, isolation and kinetic characterization of recombinant human catechol-O-methyltransferase from Pichia pastoris strains

Publication . Pedro, Augusto Quaresma Henriques; Passarinha, Luís António Paulino; Queiroz, João António de Sampaio Rodrigues

Catechol-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.

2016Doctoral thesis

Open access

Pichia pastoris: a recombinant microfactory for antibodies and human membrane proteins

Publication . Gonçalves, A M; Pedro, Augusto; Maia, C J; Sousa, Fani; Queiroz, João; Passarinha, L A

During 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.

2013-05Journal article

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