Production and single-step purification of EGFP and a biotinylated version of the Human Rhinovirus 14 3C protease

The fluorescent reporter enhanced Green Fluorescent Protein (EGFP) has been used for assaying a wide range of biological activities ranging from gene expression, or localization of target proteins through to intermolecular interactions. However, over-production of this protein in Escherichia coli has resulted in the presence of inclusion bodies, which complicates recovery of the protein in significant quantities. In this paper, we describe a single-step method for isolating the protein from a Glutathione-S-Transferase (GST) fusion protein, release of the EGFP protein from the fusion was demonstrated using a biotinylated variant of Human Rhinovirus 14 3C protease that we have also constructed. We also suggest the potential uses of the biotinylated protease for bionanotechnology and synthetic biology.