Clonagem, expressão e caracterização de lisozima de Anopheles darlingi em Pichia pastoris

Abstract

Since its discovery in 1922 by Alexander Fleming, lysozyme has become one of the most studied enzymatic models, due to its mechanism of action as a natural defense barrier against microorganisms, especially gram-positive bacteria. In nature, lysozyme is divided into 6 types: type C, type G, type I, fungal and bacterial, phages and plants. This research aims to clone and express the lysozyme of Anopheles darlingi in the methylotrophic yeast Pichia pastoris. To this end, an expression cassette was assembled for insertion of the synthetic lysozyme gene of Anopheles darlingi into the Pichia pastoris genome using the pPIC9 expression vector, confirmed by restriction analysis with the EcoRI and NotI enzymes. The expression cassette was introduced into P. pastoris by electroporation, and 40 clones resulting from the transformation were screened in minimal medium lacking amino acids. The Western Breeze chromogenic immunodetection technique confirmed 39 recombinant lysozyme-producing clones detecting the histidine tail present in the C-terminal portion in the lysozyme gene. The use of the AOX1 promoter in the expression cassette allowed the induction of the recombinant clones with 1% methanol for 96 hours in submerged fermentation and the confirmation of protein expression was performed by the electrophoretic analysis in polyacrylamide gel in denaturing condition SDS-PAGE 15% where the presence of lysozyme was detected at approximately 14 kDa. The characterization of the enzyme showed that A. darlingi lysozyme has distinct aspects regarding chicken egg lysozyme. The results obtained demonstrate the ability of Pichia pastoris yeast to express lysozyme and demonstrate the possible biotechnological potential aggregation to this research.