Expressão e caracterização de trealose-6-fosfato fosfatase de Anopheles gambiae produzida em Pichia pastoris

Abstract

Human malaria being a tropical and parasitic disease of medical, social and economic relevance represents one of the most important public health problems in the world. Given of resistance of the insect vector to insecticides, research of new tools for vector control has been proposed. The most fascinating alternative is based on the use of enzyme inhibitors that play an important and fundamental role in the metabolism and physiology of the insect. One of this enzyme is trehalose-6- phosphate phosphatase that dephosphorylates trehalose-6-phosphate substrate, forming trehalose disaccharide and inorganic phosphate. Trehalose is a dimer of glucose found in plants, insects and microorganisms. In that organisms trehalose is important to different roles, of which is to serve as a site of glucose storage for energy and/or synthesis of cellular components, another role is to protect the cells against environmental stresses such as desiccation and freezing, and to stabilize proteins against denaturation. Therefore, this study aimed to clone and express trehalose-6-phosphate phosphatase (TPP) gene from Anopheles gambiae mosquito in the methylotrophic yeast Pichia pastoris and to analyze recombinant enzyme expressed in this yeast. According to results, was demonstrated that TPP enzyme from A. gambiae has been efficiently expressed in P. pastoris and secreted into the cell culture supernatant as analyzed by Western blotting. By the electrophoretic profile in SDS-PAGE gel, after the purification procedure, has been revealed that recombinant TPP enzyme of TPP 61 clone has a molecular weight of ~ 36 kDa. The characterization of enzyme presented an optimum pH of 8.0 and optimum temperature of 38 °C. The kinetic constants of enzyme indicated a KM of 3.19 ± 0.10 mM and Vmax of 290.0 ± 3.78 μmol.min-1. The two-way ANOVA test revealed significant inhibition of recombinant TPP for EDTA (p <0.0001), NaF (p <0.0001), CaCl2 (p <0.001) and Ca(NO3)2 (p <0.0001) at 25 mM concentration, while CaCl2 (p <0.001) and Ca(NO3)2 (p <0.05) compounds were significant for inhibition enzyme at 5 mM concentration.