Expression of Soluble Recombinant Proteins via Translational Fusion to Novel IDPS

Abstract

The heterologous production of proteins in Escherichia coli cells is a common practice in basic research laboratories. However, overexpressed proteins frequently aggregate as folding intermediates in insoluble inclusion bodies thereby complicating downstream applications. While strategies have been developed to recover the aggregated proteins from inclusion bodies they are not universally effective. An alternative approach is to express heterologous proteins in the soluble form through the use of gene fusions. A variety of fusion proteins have been described for this purpose including thioredoxin (Trx), maltose binding protein (MBP), glutathione S-transferase (GST), and NusA. In this work, we present a new set of intrinsically disordered polypeptide (IDP) fusion partners that promote the soluble production of recalcitrant protein targets from E. coli. A portfolio of recalcitrant proteins was expressed in E. coli as translational fusions to N-terminal 6xHis, 6xHis-MBP, or a set of 6xHis-IDP fusions. Under a standard set of growth conditions the novel IDP-fusion proteins outperformed both the 6xHis and 6xHis-MBP fusion partners in promoting soluble expression. Furthermore, the N-terminal fusions did not inhibit affinity-purification, biological activity where tested, or fusion-tag removal by enterokinase cleavage. The higher percentage of soluble protein recovered coupled to the purification and cleavage efficiencies make these novel intrinsically disordered fusion tags attractive alternatives for protein scientists.