Clonagem e expressão do CDNA codificador do hormônio de crescimento de tambaqui (Colossoma macropomum) na levedura Pichia pastoris

Abstract

Genetic studies involving search, cloning and expression of protein-coding genes associated with important physiological processes and advantageous characteristics from an economic point of view have made biotechnological research increasingly promising. Due to their zootechnical importance, the genes encoding growth hormone (GH) from several fish species have been isolated, cloned and expressed in heterologous expression systems. Tambaqui (Colossoma macropomum), an Amazonian fish species considered promising for aquaculture, has been the subject of extensive biotechnological research aimed at expanding the species' productive capacity. In this work, the objective was to express the tambaqui GH cDNA in the methylotrophic yeast Pichia pastoris. The nucleotide sequence of the structural region of the tambaqui GH cDNA was optimized with the preferred codons of P. pastoris and obtained by chemical synthesis and subcloned into the pPIC9 expression and secretion vector. Expression was regulated by the AOX1 promoter and the recombinant protein addressed to the culture supernatant by the action of the signal peptide factor α from Saccharomyces cerevisiae. SDS-PAGE gel analysis revealed the presence of a protein band of approximately 23 kDa, which was confirmed to be recombinant tambaqui GH (rtGH) through Western blotting analysis. In the future, rtGH should be analyzed for its efficiency in accelerating the growth of tambaqui juveniles with a view to the possibility of use in aquaculture production, which will certainly represent innovation in native fish farming technologies in the Amazon.