Expressão endógena do hormônio do crescimento de tambaqui (Colossoma macropomum) em Pichia pastoris

Abstract

In the northern region, where there is the highest consumption of fish, tambaqui (Colossoma macropomum) is the most consumed fish. To enable increased production of tambaqui, it is necessary to understand the physiology, ecology, among other knowledge, for an optimization and standardization of technologies to be applied in the production of this fish. Recently, the cDNA of tambaqui growth hormone was built by chemical synthesis and expressed in P. pastoris under the control of the pAOX1 promoter of this yeast. Considering the toxicity of the inducer of this promoter, this work aims at the intracellular heterologous expression of tambaqui growth hormone (rtGH) in the yeast Pichia pastoris, using a constitutive promoter, pPGK1. Two vectors were constructed for rtGH expression, and these vectors were linearized and integrated into the genome of P. pastoris lineage M12 (auxotrophic for the amino acid leucine). The transforming clones were initially selected in the selective medium MD followed by a second selection by growth in MD medium with the antibiotic G418. To confirm the transforming colonies, the rtGH gene was identified by PCR, while the confirmation of intracellular expression of the protein was performed by immunodot and SDS-PAGE/Western Blotting. As the expression of tGH occurred at low levels, efforts should be made to identify the reason for low production, so that the expression of the hormone can be obtained at levels where recombinant yeast can be used for feed supplementation for fish.