Abstract

We developed NF8, a 35-amino acid fusion tag derived from the pentameric coiled-coil Phe-14 peptide, to enhance recombinant protein expression in Escherichia coli . NF8 significantly boosted the soluble yields of multiple challenging proteins by 1.9–77.6-fold, including the PET hydrolase LCC-WCCG. Notably, while LCC-WCCG is typically expressed at low temperatures (16–25 °C) to maintain solubility, the NF8 system enabled its high-level expression at 30 °C without compromising enzymatic activity. The tag promotes pentamerization and confers proteolytic resistance, thereby improving protein stability. Unlike larger tags, NF8 minimally impacts protein function, and its removal does not alter enzymatic activity. Specifically, NF8-tagged LCC-WCCG exhibited 60% higher PET degradation activity in crude lysates, with significant potential for advancing enzymatic plastic waste recycling. It was found that NF8 outperformed common tags (SUMO, GST, NusA) in both expression and functional preservation. Therefore, NF8 is a robust system for high-yield production of functional proteins, especially those that do not require complex multimeric assembly.