Mutação e subclonagem do gene PaOLP em vetor de expressão em Pichia pastoris.

Abstract

The protein expression in heterologous systems represents a power tool in order to produce in large scale important proteins, aiming to demonstrate their activity or biotechnological applications. In our laboratory it was isolated a PR5-like gene from Physalis angulata that encodes an antifungal protein, denominated PaOLP. In this work it was performed the subcloning of the PaOLP gene, from pGEM-T Easy cloning vector to the pPICZa-A Pichia pastoris expression vector. The PaOLP gene was mutated by PCR to delete regions encoding the signal and carboxi-termini peptides, and cloned into Escherichia co/i strain DH5a cells, which were zeocina selected and positives for colony PCR using PPM7 and PPM8 primers, and by digestion with appropriate enzymes. The used sub-cloning approach may lead to expression of a mature recombinant PaOLP protein, with sorting to the extracellular medium of Pichia pastoris yeast cells and possessing an appropriated His-tag to purification on immobilized methal a:ffinity cbromatography. The introduction of the pPICZa-A vector leading the mutated PaOLP gene into the Pichia pastoris competent cells, and posterior gene expression to in large scale production of the fusion mature PaOLP protein, are the current subjects o f this research.