Membrane-Bound Catechol-O-Methyltransferase Biosynthesis in Pichia pastoris: Optimization of the Induction Phase Using Artificial Neural Networks

摘要

Membrane proteins are drug targets for a wide range of diseases and the achievement of appropriate samples for further research encourages the pharmaceutical industry for developing new and more effective drugs. These samples are often obtained toward a strategy concerning the biosynthesis of the target protein on a large scale, after which an equal appropriated purification process has to be designed in order to isolate pure protein in higher quantities than those found in the protein's native source. The 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 membrane-bound catechol-O-methyltransferase are still hampered by its low recombinant production, Pichia pastoris has been described as an attractive host for the production of correctly folded and inserted membrane proteins [1, 2]. Hence, in this work and for the first time, recombinant membrane-bound catechol-O-methyltransferase biosynthesis was carried out in mini-biorreactors (working volume 1L) using Pichia pastoris in modified basal salts medium containing methanol. Preliminary results indicated that several independent variables associated with the methanol induction phase such as the temperature (20 to 30 °C), the methanol concentration (1 to 3 mL/L/h) and the addition of dimethylsulfoxide (4 to 6 % (v/v)) greatly influence the production levels of the target protein. Therefore, these three independent conditions were selected for optimization as independent process parameters and the optimization aim was to achieve a maximum membrane-bound catechol-O-methyltransferase biological activity. Then, predictive artificial neural network models of the membrane-bound catechol-O-methyltransferase biological activity as function of the selected inputs were proposed based on these exploratory experiments. In conclusion, for the first time, recombinant membrane-bound catechol-O-methyltransferase biosynthesis was successfully attained in Pichia pastoris at a bioreactor-scale in which the optimization of the induction phase led to a great improvement in the strategies currently applied for the production of this protein.