An Intrinsically Disordered Peptide Tag that Confers an Unusual Solubility to Aggregation-Prone Proteins

Abstract

There is a high demand for the production of recombinant proteins in Escherichia coli for biotechnological applications, but their production is still limited by their insolubility. Fusion tags have been successfully used to enhance the solubility of aggregation-prone proteins; however, smaller and more powerful tags are desired for increasing the yield and quality of target proteins. Here, the NEXT tag, a 53-amino-acid-long solubility enhancer, is described. The NEXT tag showed outstanding ability to improve both in vivo and in vitro solubilities, with minimal effect on passenger proteins. The C-terminal region of the tag was mostly responsible for in vitro solubility, while the N-terminal region was essential for in vivo soluble expression. The NEXT tag appeared to be intrinsically disordered and seemed to exclude neighboring molecules and prevent protein aggregation by acting as an entropic bristle. This novel peptide tag should have general use as a fusion partner to increase the yield and quality of difficult-to-express proteins. IMPORTANCE Production of recombinant proteins in Escherichia coli still suffers from the insolubility problem. Conventional solubility enhancers with large sizes, represented by maltose-binding protein (MBP), have remained the first-choice tags; however, the success of the soluble expression of tagged proteins is largely unpredictable. In addition, the large tags can negatively affect the function of target proteins. In this work, the NEXT tag, an intrinsically disordered peptide, was introduced as a small but powerful alternative to MBP. The NEXT tag could significantly improve both the expression level and the solubility of target proteins, including a thermostable carbonic anhydrase and a polyethylene terephthalate (PET)-degrading enzyme that are remarkable enzymes for environmental bioremediation.