Purification of the GfsA-3x FLAG Protein Expressed in Aspergillus nidulans

Abstract

GfsA is a fungal β-galactofuranosyltransferase involved in the biosynthesis of O-glycan. To investigate the enzymatic functions of GfsA, we attempted to obtain a recombinant protein of this enzyme from two heterologous host organisms. However, GfsA could not be expressed as a recombinant protein in either Escherichia coli (E. coli) or Saccharomyces cerevisiae (S. cerevisiae). Therefore, we decided to employ Aspergillus nidulans (A. nidulans) as the host organism, and produced a strain that expressed 3x FLAG-tagged GfsA using chromosomal tagging. To confirm its expression, a solubilized protein was prepared from the tagged strain and analyzed with an anti-FLAG antibody. The strain that expressed 3x FLAG-tagged GfsA produced a functional protein with a mass of approximately 67 kDa. The method described in this manuscript allows purification of the GfsA-3xFLAG protein as expressed in A. nidulans cells.