Research Article: Expression and localization of the enhanced green fluorescent protein in the peroxisomes of the methylotrophic yeast Pichia pastoris

Abstract

Abstract The enhanced green fluorescent protein (EGFP) is a macromolecule which fluoresces green under specific wavelengths of light. It has been widely used as a tool to study cell structure and function. EGFP-PTS1 is a fusion of the EGFP gene and a peroxisome targeting sequence (PTS1). Attachment of the PTS1 localizes EGFP to the peroxisome, an organelle found in eukaryotic cells. In this work, DNA encoding an EGFP-PTS1 fusion was inserted into a plasmid which contained a selectable marker gene for adenine biosynthesis (ADE1). The plasmid, called pCO1, was transformed into an adenine auxotrophic strain (ade1) of the yeast Pichia pastoris. The ade1 strain, which is pink in color, could not synthesize its own adenine and thus could not survive without this nitrogenous base in its growth medium. The ADE1 gene on pCO1 acted as a form of selection to identify cells transformed by the plasmid. Colonies transformed by pCO1 were white in color and could grow in medium without the supplemental addition of adenin...